Prostate cancer-specific alterations in erg gene expression and detection and treatment methods based on those alterations

ABSTRACT

Alterations in ERG gene expression can be observed in patients with prostate cancer. Specific ERG isoforms are associated with, or involved in, prostate cancer. Compositions comprising these isoforms provide therapeutic benefit and can be used in methods of detecting, diagnosing, prognosing, and treating prostate cancer. These compositions provide biomarkers for detecting the expression of combinations of the PSA/KLK3, PMEPA1, NKX3.1, ODC1, AMD1 and ERG genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/081,101filed Apr. 10, 2008 (U.S. Pat. No. 9,206,481), which is aContinuation-in-Part of PCT/US2007/080828, filed Oct. 9, 2007, which inturn claims the benefit of U.S. provisional application Nos. 60/929,505,filed Jun. 29, 2007, and 60/850,254, filed Oct. 10, 2006. The entiredisclosure of each of these applications is relied upon and incorporatedby reference.

GOVERNMENT LICENSE RIGHTS

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant R01DK065977 awarded by the National institutes of Health.

TECHNICAL FIELD

The invention relates to polynucleotide and polypeptide sequences thatare involved in, or associated with, prostate cancer. The inventionfurther relates to therapeutic compositions and to methods of detecting,diagnosing, and treating prostate cancer.

BACKGROUND

ETS Related Gene (ERG), a member of the ETS transcription family, wasinitially isolated and described in 1987 (Reddy et al., PROC. NATL.ACAD. SCI. USA 84:6131-35 (1987) Rao et al., SCIENCE 237:635-39 (1987)).Like other members of the ETS family, it plays a central role inmediating mitogenic signals transmitted by major cellular pathways,including the MAPK pathway. Proteins in the ETS family show a widevariety of expression patterns in human tissues. ERG is expressed inendothelial tissues, hematopoietic cells, kidney, and in the urogenitaltrack. (Oikawa et al., GENE 303:11-34 (2003).) Expression of ERG hasalso been detected in endothelial cells (microvessels) of the stroma ina small proportion of prostate cancer. (Gavrilov et al., EUR CANCER 37:033-40 (2001).)

The ERG protein participates in the regulation of gene expression bybinding both to DNA comprising a 5′-GGA(A/T)-3′ consensus sequence andto the Jun/Fos heterodimer. These interactions occur via the highlyconserved ETS domain. (Verger et al., J BIOL CHEM 276: 17181-89 (2001).)Splice variants exist, and of the nine that have been reported, ERG6 andERG9 have multiple stop codons that likely render them non-functional.(Owczarek et al., GENE 324: 65-77 (2004).) ERG7 and ERG8 can bedistinguished from ERG1-5 by the absence of exon 16, (Id.) In addition,the ERG8 transcript is unique in its inclusion of a 3′ sequencefollowing exon 12, a portion of which forms part of the open readingframe. (Id.) ERG8 had been previously described as a 1460 base pairlinear mRNA, with the National Center for Biotechnology information(“NCBI”) Accession No. AY204742 (Owczarek et al., (2004).)

ERG, like other members of the ETS family, is a proto-oncogene withtransforming activity. (Oikawa et al., GENE 303:11-34 (2003); Hsu etal., J CELL BIOCHEM 91:896-903 (2004); Reddy et al., PROC NATL ACAD SCIUSA, 84:6131-35 (1987); Hart et al., ONCOGENE 10:1423-30 (1995);Sementchenko et al., ONCOGENE 17:2883-88 (1998) Chromosomaltranslocations involving ERG have been linked to Ewing sarcoma, myeloidleukemia, and cervical carcinoma. (Oikawa et al., GENE 303:11-34(2003).) It has recently been shown that ERG1 is the most commonlyoverexpressed proto-oncogene in malignant prostatic tissue. (Petrovicset al., ONCOGENE 24:3847-52 (2005).) Independently, Tomlins et al.,SCIENCE 310:644-48 (2005), described novel gene fusions involving ERGand TMPRSS2, are androgen-sensitive gene, that may provide at least onepossible mechanism for ERG1 overexpression. At least two additionalstudies have confirmed ERG rearrangements in prostate cancer. (Soller etal., GENES CHROMOSOMES CANCER 45:717-19 (2006); Yoshimote et al.,NEOPLASIA 8:465-69 (2006).)

Although prostate cancer is the most common non-skin cancer in NorthAmerican men and the third leading cause of cancer mortality (Jemal etal., CANCER J CLIN 56:106-30 (2005)) remarkably little is known aboutcritical events in prostatic carcinogenesis. While recent reports ofhigh frequency genomic rearrangements involving the ERG locus and ERG1overexpression are intriguing there remains a need in the art toidentify and characterize the gene expression products of the ERG locusin prostate cancer. Cancer-derived transcripts, splice varianttranscripts, and altered expression ratios between transcripts arehighly specific tools that can be used for cancer diagnosis throughoutthe different stages of cancer development. In addition, targetedinhibition or activation of these products, and/or direct manipulationof cancer-specific promoters, can be used as highly selectivetherapeutic strategies to target the causative root of cancer. Thus, theidentification of molecular alterations specific for prostate cancerwould not only permit optimization of diagnosis and prognosis but alsowould permit establishment of individualized treatments tailored to themolecular profile of the tumor.

In addition, while prostate cancer is increasingly detected early, theprognosis of individual patients remains a challenge. Identification ofmolecular biomarkers representing functionally relevant pathways thatcan distinguish between aggressive and indolent forms of prostate cancerat early stages will have tremendous impact in improving prognostic andtherapeutic decisions. Other than serum PSA, currently there are norational (tumor biology based) prognostic or therapeutic molecularbiomarkers available in the clinical practice of prostate cancer.

While 80% of prostate cancer patients respond well to surgery, radiationtherapy or watchful waiting, about 20% will develop metastasis that isoften fatal to patients. Initially, prostate cancer development isdriven by the androgen receptor (AR) pathway. (Heinlein et al.,ENDOCRINE REV 25:276-308 (2004); Linja et al., J STEROID BIOCHEM MOLBIOL. 92: 255-64 (2004); Shaffer et al., LANCET ONCOL. 4:407-14 (2003);Chen et al., NAT MED 10: 26-7 (2004).) However, frequent alterations ofAR structure and/or function are well recognized during prostate cancerprogression especially with metastatic disease. Other genetic pathwaysthat are often altered in these late stage androgen-independent tumorsinclude p53 mutations. BCL2 overexpression and mutations or reducedexpression of PTEN. (Shaffer et al., LANCET ONCOL 4:407-14 (2003).)Importantly, both p53 and PTEN pathways may affect AR functions.

Defects in AR-mediated signaling are increasingly highlighted forpotential causal roles in prostate cancer progression. (Heinlein et al.,ENDOCRINE REV 25:276-308 (2004); Dehm et al., J CELL BIOCHEM 99: 333-344(2006).) Prostate cancer associated alterations of AR functions byvarious mechanisms, including AR mutations, AR gene amplification,altered AR mRNA or AR protein levels, changes in AR interaction withco-activators/co-repressors and ligand independent AR activation bygrowth factors/cytokines, may all contribute to prostate cancerprogression. (Gelmamn, J CLIN ONCOL 20:3001-15 (2002); Grossman et al.,J NATL CANCER INST 93: 1687-97 (2001).) Due to the lack of preciseknowledge of AR dysfunctions in pathologic specimens, it is difficult toidentify patients with function& defects of AR.

The choice of therapy for late stage prostate cancer is systemicandrogen ablation, which eventually fails in most patients. Therefore,the knowledge of AR pathway dysfunctions that are predictive of androgenablation therapy failure would significantly impact the patientstratification for new emerging therapeutic strategies.

Unlike in breast cancer where estrogen receptor protein status inprimary tumor is effectively used in making therapeutic and prognosticdecisions (Yamashita et al., BREAST CANCER 13(1):74-83 (2006); Martinezet al., J SURG 191(2):281 (2006); Giacinti et al., ONCOLOGIST 11(1):1-8(2006); Regan et al., BREAST 14(6):582-93(2005); Singh et al., J CELLBIOCHEM 96(3):490-505 (2005)), AR protein expression status does notappear to be useful in prostate cancer, likely because many factorsbesides AR protein expression level may affect AR activity. Although ARexpression can be detected throughout the progression of prostatecancer, it is heterogeneous and changes over time. Several studies haveindicated that AR expression is reduced in poorly differentiated areaswith a higher Gleason score. (Heinlein et al., ENDOCRINE REV 25:276-308(2004); Linja et al., J STEROID BIOCHEM MOL BIOL 92: 255-64 (2004);Shaffer et al., LANCET ONCOL 4:407-14 (2003); Chen et al., NAT MED 10:26-7 (2004); Gelmann, CLIN ONCOL 20:3001-15 (2002); Grossman et al., JNATL. CANCER INST 93:1687-97 (2001); Krishnan et al., CLIN CANCER RES6:1922-30 (2000).)

In contrast, some recent reports found that higher AR expression isassociated with higher clinical stage, higher Gleason score, and withdecreased PSA recurrence-free survival. (Linja et al., CANCER RES61:3550-55 (2001); Sweat et al., J UROL 161:1229-32 (1999); Li et al.,AM J SURG PATHOL 28:928-34 (2004).) Part of the reason for thiscontroversy is the inherent heterogeneity of AR expression in theprostate and the semi-quantitative nature of immunohistochemicalevaluations. (Krishnan et al., CLIN CANCER RES 8:1922-30 (2000).) Inrecent years, our laboratory has established novel insights into theandrogen regulated transcriptome and identified AR targets which havepromise in defining the role of AR dysfunctions in prostate cancer, aswell as in providing novel biology based biomarkers and therapeutictargets during prostate cancer progression. (Xu et al., CANCER RES.63(15):4299-304 (2003); Segawa et al., ONCOGENE 21(57):8749-58 (2002);Xu et al., INT J CANCER 92(3):322-8 (2001); Xu et al., GENOMICS 66(3):257-263 (2000); Masuda et al., J MOL BIOL 353(4):763-71 (2005); Richteret al., PROSTATE CANCER PROSTATIC DIS 10(2):114-8 (2007).

Nevertheless, a need still exists to streamline the functionalevaluation of AR defects at early stages of prostate cancer, when theimpact of this knowledge on disease management will be more profound.The present application meets this need by providing a read out for themeasurement of the expression of carefully selected AR downstreamtargets. This read out provides information on the in viva functionalstatus of AR in prostate cancer cells, which helps to stratify patientsbased on AR signal amplitude and can be used to help prognose prostatecancer and provide new ways of managing and treating these patients.

In particular, a need exists to further characterize the ERG8protooncogene and its role in prostate cancer. ERG8 provides an untappedsource of diagnostic, prognostic, and therapeutic agents applicable toprostate cancer

Citation of references herein shall not be construed as an admissionthat such references are prior art to the present invention.

SUMMARY

Transcription of the ERG gene is altered in prostate cancer cellscompared to benign cells. The present application describes for thefirst time the complete ERG8 nucleotide sequence and also describes thepredominant expression of the ERG8 isoform in cancerous cells. It alsoprovides the sequence and characterization of two unique,cancer-specific transcripts of the ERG locus, ERG ProstateCancer-specific Isoform 1 (EPC1) and EPC2. The disclosed ERG isoformscan be used alone or in combination as biomarkers of prostate cancer, astargets for therapeutic intervention, or to develop therapeutic agents.In addition, the disclosure describes a novel, prostate cancer-specificERG promoter. The ERG promoter can be used to selectively targetexpression of therapeutic proteins, such as cellular toxins, to prostatecancer cells. Polynucleotide transcripts produced from this novelpromoter can also be detected as biomarkers for prostate cancerdiagnosis, or to aid in prognosis of prostate cancer.

In one aspect, the disclosure provides the nucleic acid sequences andencoded protein sequences for cancer-specific gene transcripts of theERG locus, including ERG8, EPC1, and EPC2. Antibodies to the encodedpolypeptides, and to fragments of those polypeptides, are alsodescribed. In some embodiments, the antibody binds an epitope of thepolypeptide or polypeptide fragment that is linear, whereas in otherembodiments the epitope is conformational. In some embodiments, theepitope is contained within, or comprising, the unique carboxy-terminusof the EPC1 or EPC2 polypeptide. Some of the antibodies that bind anepitope in the carboxy terminus of EPC1 or EPC2 also bind the respectiveEPC1 or EPC2 polypeptide.

The disclosure further provides kits for detecting prostate cancer.These kits can be used to detect (either qualitatively orquantitatively) nucleic acids or proteins that serve as prostate cancermarkers. For example, the expression of prostate cancer-specificisoforms of the ERG gene, such as ERG8, EPC1, EPC2, or the transcriptsproduced by the prostate cancer-specific promoter, when detected in abiological sample from a subject, either alone or in combination withother cancer markers, can be used to indicate the presence of prostatecancer in the subject or a higher predisposition of the subject todevelop prostate cancer, or they can be used to predict the severity orstage of prostate cancer, such as whether the cancer is high risk or amoderate risk cancer.

In some embodiments, the kits comprise a nucleic acid probe, such as theprobes described elsewhere in the disclosure, that hybridizes underdefined conditions to an ERG sequence. The nucleic acid probe canhybridize SEQ ID NO: 1 (ERG8). SEQ ID NO: 3 (EPC1), SEQ ID NO: 5 (EPC2),SEQ ID NO: 30 (ERG8), or SEQ ID NO: 46 (ERG8) (or sequencescomplimentary to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:30, or SEQ ID NO: 46), or a combination of probes can be used tohybridize to ERG8 and EPC1, ERG8 and EPC2, EPC1 and EPC2, or even ERG8,EPC1, and EPC2. In other embodiments, the kits comprise first and secondoligonucleotide primers that hybridize to non-overlapping sequences inERG8 (SEQ ID NOS: 1, 30, and 46), EPC1 (SEQ ID NO: 3), or EPC2 (SEQ IDNO: 5). In some embodiments, primer pairs that hybridize to ERG8 andEPC1; ERG8 and EPC2; EPC1 and EPC2; ERG8, EPC1, and EPC2, are used incombination. In such cases, one or more of the ERG8, EPC1, or EPC2primers may be the same.

The disclosure additionally describes diagnostic kits comprising ananti-ERG isoform-specific antibody, for example, an anti-ERG8 antibody,an anti-EPC1 antibody, or anti-EPC2 antibody. In one embodiment, thedisclosure provides an anti-EPC1 antibody that binds an epitopecomprising acids amino acids 217 to 220 of SEQ ID NO: 4. In anotherembodiment, the antibody is an anti-EPC2 antibody that binds an epitopewithin or comprising amino acids 28 to 97 of SEQ ID NO: 6. In each case,the epitope can be a linear epitope or a conformational epitope. In someembodiments, combinations of antibodies can be included in the kit. Forexample, a kit can comprise anti-ERG8 and anti-EPC1 antibodies,anti-ERG8 and anti-EPC2 antibodies, anti-EPC1 and anti-EPC2 antibodies,or anti-ERG8, anti-EPC1, and anti-EPC2 antibodies. The antibodies canbe, optionally, detectably labeled.

ERG isoform expression can be used to diagnose or prognose prostatecancer. The disclosure therefore also provides methods for detecting theexpression of one or more of ERG8, EPC1, or EPC2 in a biological sample,such as prostate tissue, blood, serum, plasma, urine, saliva, orprostatic fluid. For example, in some embodiments, the methods comprisedetecting amplification products of ERG8, EPC1, or EPC2 usinghybridization-based techniques. In other embodiments, amplificationproducts are size separated and visualized as part of the detectionmethods. The methods of diagnosing or prognosing prostate cancer canfurther comprise measuring the expression level (e.g. mRNA orpolypeptide) of ERG8, EPC1, or EPC2, anal correlating the expressionlevel of the ERG isoform with the presence of prostate cancer or ahigher predisposition to develop prostate cancer in the subject, or withthe severity or stage of prostate cancer, such as high risk or moderaterisk prostate cancer.

In some embodiments, the methods comprise detecting the expression ofthe ERG8 isoform. In other embodiments, it is the expression of the EPC1isoform that is detected. In yet other embodiments, the EPC2 isoform isdetected. In still other embodiments, the methods comprise detecting theERG8 and EPC1 isoforms in combination, the ERG8 and EPC2 isoforms incombination, the EPC1 and EPC2 isoforms in combination, or thecombination of the ERG8, EPC1, and EPC2 isoforms. In each case, each ERGisoform can be detected and/or measured by detecting and/or measuringthe transcript, or by detecting and/or measuring the correspondingpolypeptide.

Therapeutic methods of treating prostate cancer and treating disordersof prostate hyperproliferation are also disclosed. For example, thedisclosure provides methods of treating prostate cancer comprisingdestabilizing a prostate cancer-specific ERG gene transcript in prostatecancer cells. In some embodiments, the methods comprise destabilizingone, all, or any combination of ERG8, EPC1, EPC2, ERG1, ERG2, and/orERG3 transcripts, resulting in degradation of those transcripts andinhibition of expression of the encoded polypeptide(s). In oneembodiment, the destabilization employs siRNA. In another embodiment,the methods employ small hairpin RNAs (shRNA). In yet anotherembodiment, an antisense molecule is used to destabilize thetranscript(s). In still another embodiment, a ribozyme is used to causedestabilization. Small molecule inhibitors can also be used to inhibitexpression of one or more ERG isoforms. The disclosure also providesmethods of using an antibody to one or more ERG is to treat prostatecancer or disorders of prostate hyperproliferation. Thus, in varyingembodiments the disclosure provides methods of treating prostate canceror disorders of prostate hyperproliferation comprising administering ananti-ERG8, an anti-EPC1, an anti-EPC2, an anti-E 1, and anti-ERG2, ananti-ERG3 antibody, or a combination of those antibodies. In someembodiments, a single antibody may be specific for one or more proteinsencoded by the disclosed ERG isoforms.

In another embodiment, the present application provides a panel ofbiomarkers for prostate cancer, methods and systems for using thosebiomarkers to diagnose and prognose prostate cancer, and diagnostic andprognostic kits comprising reagents used to detect the biomarkers. Inone embodiment the panel comprises a combination of two or more of a setof six androgen inducible/co-regulated genes (PSA/KLK3, PMEPA1, NKX3.1,ODC1, AMD1, and ERG). In some embodiments, the ERG gene is EPC1, EPC2,ERG1, ERG2, ERG3, ERG8, or combinations thereof.

The present application also provides prognostic kits that detect ormeasure the levels of two or more androgen inducible/co-regulated genes.The prognostic kits are used in methods of predicting the functionalstatus of in vivo androgen receptor signaling or in methods ofpredicting prostate cancer progression or severity, such as predictingwhether the prostate cancer is a moderate risk prostate cancer or a highrisk prostate cancer, predicting the prostate cancer stage (e.g., usingthe T staging system (pTX, pT0, PT1, pT2, pT3, pT4) or theWhitmore-Jewett system (A, B, C, D)), or predicting whether the prostatecancer is progressing, regressing, or in remission. The prognostic kitscan also be used to predict disease-free survival followingprostatectomy, which can be defined, for example, by serum PSA levelequal or higher than 0.2 ng/ml after prostatectomy. In some embodiments,the prognostic panel comprises two or more of the following genes:PSA/KLK3, PMEPA1, NKX3.1, ODC1, AMD1, and ERG. In certain embodiments,the ERG gene is EPC1, EPC2, ERG1, ERG2, ERG3, ERG8, or combinationsthereof. Accordingly, assays using the prognostic kits can detect ormeasure the levels of two or more of these genes. For example, aprognostic kit can be used to measure the levels of two, three, four,five, six, or even more androgen inducible/co-regulated genes.

In certain embodiments, the prognostic assay further comprises detectingor measuring PSA, % PSA, PSA doubling time, PSA velocity, prostatevolume or a combination of these indicators.

In prognostic embodiments, the method of prognosing prostate cancer cancomprise detecting or measuring in a biological sample from anindividual the expression of two or more of genes chosen from PSA/KLK3,PMEPA1, NKX3.1, ODC1, AMD1, and ERG; and comparing, for the expressionof each gene detected or measured, the results obtained in (a) with theexpression of the same gene in a control sample.

In a prognostic method, the altered expression of the two or more genesin the patient sample relative to the control sample is predictive ofdisease severity, for example a moderate risk prostate cancer or a highrisk prostate cancer. The altered expression may also be predictive ofwhether the prostate cancer is progressing, regressing, or in remission.Alternatively, a threshold value of gene expression can be selected andused as the control sample. In this case, if the gene expression levelis less n the threshold value, it is considered reduced. The thresholdvalue can be determined using known techniques. For example, the valuecan be determined from the mRNA copy number or the cycle thresholdvalue.

Although increases and decreases of at least 10% relative to a controlor threshold value can be used in the prognostic methods, other valuesmay also be used. For example, the increase or decrease may be at least20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or even 500%. Theincrease or decrease may also be expressed in terms of statisticalsignificance, where a statistically significant increase or decrease inexpression, such as p<0.06, p<0.01, p<0.005, or p<0.001, indicates thepresence of prostate cancer or a higher predisposition to developprostate cancer, prostate cancer progression, or disease severity.

In some prognostic embodiments, a decrease in expression levels of theandrogen inducible/co-regulated gene(s) is used to predict compromisedandrogen receptor signaling, which in turn is predictive of the presenceor predisposition to develop high risk or advanced stage prostate canceror a reduced disease-free survival time following prostatectomy.

The disclosure also provides methods of detecting the expression of twoor more of PSA/KLK3, PMEPA1, NKX3.1, ODC1, AMD1, and ERG (includingEPC1, EPC2, ERG1, ERG2, ERG3 or ERG8) in a biological sample, such asprostate tissue or a biofluid, such as, blood, serum, plasma, urine,saliva, or prostatic fluid. For example, in some embodiments, themethods comprise detecting amplification products of PSA/KLK3, PMEPA1,NKX3.1, ODC1, AMD1, and ERG using hybridization-based techniques. Inother embodiments, amplification products are size separated andvisualized as part of the detection methods. The methods of prognosingprostate cancer can also comprise measuring the expression level of theproteins encoded by PSA/KLK3, PMEPA1, NKX3.1, ODC1, AMD1, and ERG, forexample by using an antibody.

Additional objects will be set forth in part in the description thatfollows, and in part will be understood from the description, or may belearned by practice of the invention. It is to be understood that boththe foregoing general description and the following detailed descriptionare exemplary and explanatory only and are not restrictive of theinvention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains two drawings, FIGS. 20 and 21, which areexecuted in color. Copies of this application publication with colordrawings will be provided by U.S. Patent and Trademark Office uponrequest and payment of the necessary fee.

FIGS. 1A-F show the nucleotide sequence of the complete ERG8 gene (SEQID NO: 30), aligned with the partial gene sequence of NCBI AY204742 (SEQID NO: 62).

FIG. 2 presents PCR amplification gels of ERG1, ERG2, ERG3, and ERG8transcripts in normal prostate tissue (NP) and in the prostate cancercell line VCaP.

FIG. 3 shows the PCR amplification results for ERG8 transcriptexpression n tumor cells (T) and benign epithelial cells (N) from eightpatients.

FIG. 4 shows the PCR amplification results for EPC1 transcriptexpression in tumor cells (T) and benign epithelial cells (N) from fivepatients.

FIG. 5A present a schematic diagram of the primer positions for EPC1,ERG8, and ERG1&2 specific primers. FIG. 5B presents the copy numbers ofERG1&2, ERG8, and EPC1 in VCaP cells. FIG. 5C presents the copy numbersof ERG8 and ERG1&2 using microdissected tumor cells from ten prostatecancer patients.

FIG. 6 shows a map of alternative transcription start sites in ERG exon9 (nucleotides 486 to 532 of SEQ ID NO: 7).

FIG. 7 plots the ability of three segments of the prostatecancer-specific ERG promoter to support expression of a luciferasereport construct in the VCaP cell line in comparison to the LNCaP cellline.

FIG. 8 provides the results of a Pearson correlation analysis ofTMPRSS2-ERG fusion A transcript expression with ERG1, AR, PSA, PMEPA1and LTF expression in tumor tissue.

FIG. 9A shows that downregulating ERG increases the expression ofandrogen receptor responsive genes. The top panel shows a geldemonstrating that inhibition of ERG with two different siRNAs resultsin increased expression of androgen-inducible PSA and NKX3.1transcripts. The bottom panel shows that PSA levels also increase in theculture supernatant of VCaP cells when ERG is inhibited with siRNAs.

FIG. 9B shows that ERG knockdown inhibits prostate tumor cell growthboth in vitro and in an in vivo SCID mouse tumorigenicity assay. The topleft panel shows the morphology of VCaP cells transfected with 50 nM ERGsiRNA or control (“NT”) RNA. The top right panel shows the inhibitoryeffect of ERG siRNA (dark grey bars) on VCaP cell proliferation,compared to control RNA (light grey bars). The bottom left panel shows acell cycle analysis demonstrating the inhibitory effect of ERG siRNA(dark grey bars) on the number of cells in S phase, compared to controlRNA (light grey bars). The table shows a redistribution of the number ofcells in G1, S and G2+M phases as a result of ERG siRNA treatment,measured by FACS analysis. The bottom right panel shows the inhibitoryeffect of ERG siRNA (dark grey bars) on in vivo tumor volume, comparedto control RNA (light grey bars).

FIG. 9C demonstrates that the transfection efficiency of VCaP cellstransfected with 50 nM of ERG siGLO or NT siGLO (both from DharmaconResearch, Lafayette, Colo.) and cultured for two days was nearly 100%.

FIG. 10 is a diagram showing that ERG expression can result ininhibition of the androgen receptor responsive genes PSA and NKX3.1,thereby inhibiting cellular differentiation.

FIG. 11 shows the results of siRNA inhibition of ERG expression in VCaPprostate cancer cells. Panel A shows a microscope field of control VCaPcells and Panel B shows a microscope field of cells treated with siRNA-1(SEQ ID NO: 28).

FIG. 12 compares the intensity of gene expression of the androgenregulated genes PSA/KLK3, NKX3.1, PMEPA1, ODC1, AMD1, and ERG in tumorand matched benign cells from 40 prostate cancer patients. Z-scorenormalized GeneChip derived expression intensities are depicted by heatmaps on a high-to-low scale after hierarchical clustering. Patientnumbers (N=40) are listed above the heat map. Matched tumor and benignspecimens are listed in the same order.

FIG. 13 shows a heat map display comparing the intensity of geneexpression of the prostate cancer related genes ERG, AMACR, DD3, PSGR,and PGMEM1 in cells microdissected from prostate tissue sections.

FIG. 14 shows the correlation of androgen regulated PSA/KLK3 and PMEPA1genes with ERG expression in tumor cells of prostate cancer patientsharboring TMPRSS2-ERG fusion using QRT-PCR.

FIG. 15 demonstrates that ERG expression mirrors androgen signaling inprostate cancer tissue. TMPRSS2-ERG fusion (left panel) and RSA/KLK3(right panel) transcript levels were compared in prostate cancer cellsof pT3 and pT2 stage tumors by quantitative PCR. Y-axis scales representfold changes of tissue expression levels relative to the expression ofthe GAPDH housekeeping gene.

FIG. 16 shows the distribution of biochemical recurrence and tissuePSA/KLK3 mRNA expression in tumor cells of prostate cancer (CaP)patients. Relative expression of PSA/KLK3 mRNA in tumor cells,represented by vertical bars, is shown on a log 2 scale. Darkened barsindicate patients with biochemical recurrence.

FIG. 17 shows a Kaplan-Meier survival estimation curve for time to PSArecurrence-free survival by tumor tissue PSA/KLK3 mRNA quintiles amongpatients with serum PSA 2-10 ng/ml. Quintiles are presented indecreasing order with quintile 1 referring to the highest and quintile 5to the lowest PSA/KLK3 expression (N=79). Lower tissue PSA/KLK3 mRNAexpression in prostate tumor cells correlates with an increased risk ofbiochemical recurrence.

FIG. 18 shows the activation of the oncogene C-MYC by ERG. The leftpanel shows the result of RT-PCR analysis of VCaP cells treated with ERGsiRNA. The top right panel shows the morphology of the VCaP cells aftereight days of treatment with control (“NT”), ERG siRNA, MYC siRNA, andboth ERG siRNA and MYC siRNA. The bottom right panel shows a Westernblot analysis of the effect of ERG siRNA on C-MYC expression in VCaPcells. It also shows the correlation between ERG expression and MYCexpression in microdissected human prostate tumors.

FIG. 19 shows the effect of ERG siRNA on the density and morphology ofVCaP cells, compared to NT control cells.

FIG. 20 shows a gene network in ERG-expressing human prostate tumors.Seven well-differentiated prostate tumors overexpressing ERG wereanalyzed with Bibliosphere software. Red (medium grey) and yellow (lightgrey) boxes indicate upregulation, shades of blue (dark grey) indicatedownregulation.

FIG. 21 shows a gene network affected response to ERG knockdown in VCaPcells.

FIG. 22 shows Western blots demonstrating diminishing PSA protein levelsand decreased recruitment of AR to the PSA AREIII enhancer in responseto transient ERG expression.

Panels A and B in FIG. 22 show VCaP and LNCaP cells, respectively,infected with adenoviral ERG (“Ad-ERG”) or adenoviral control(“Control”) vectors. Cell lysates prepared at 24, 48, and 72 hourspost-infection were analyzed by immunoblot using anti-ERG, anti-PSA, andanti-tubulin antibodies.

Panels C and D in FIG. 22 show ChIP assessment of AR recruitment to theKLK3/PSA gene AREIII enhancer in VCaP and LNCaP cells in response to thetransient expression of ERG by adenoviral Ad-ERG or Control vectors.“Input” indicates control genomic DNA amplicons.

FIG. 23 shows the repression of prostate differentiation genes by ERG.The top left panel shows the increase in PSA mRNA expression resultingfrom ERG siRNA transfection of VCaP cells, measured by QRT-PCR. The tomiddle panel shows a corresponding increase in PSA protein expression,measured by Western blot. The bottom left panel shows increased ARbinding to the PSA enhancer (“ARE”) and decreased ERG recruitment to theoverlapping ETS cognate element, measured by ChIP assay. The right panelshows an immunofluorescence micrograph of VCaP cells nine days aftertreatment with ERG siRNA. The cells were stained with antibodies tocytokeratin (“CK8/18”) or PSA, or stained for DNA; the right panelcompares merged images. The scale bar represents 25 microns.

FIG. 24 shows the increase in prostein (“SLC45A3”) expression in VCaPcells resulting from transfection with ERG siRNA. The top left panelshows a Western blot with antibodies to SLC45A3 and tubulin (control).The bottom let panel assesses recruitment of AR and ERG to the SLC45A3promoter upstream ARE and ETS elements by ChIP assay. The right panelshows a matrix of immunostaining results from 26 prostate tumorsexamined with an antibody to SLC45A3. “CN” indicates the case number ofthe tumor tissue. “TM-ERG” indicates the presence (black bar) or absence(white bar) of the TMPRSS2-ERG gene fusion in the tumor. “SLC” indicatesstrong (dark grey bars) or weak (light grey bars) immunohistochemicalstaining with an antibody to SLC45A3.

DETAILED DESCRIPTION Definitions

The term “ERG” refers to the ERG gene, as well as to the various ERGcDNAs and mRNAs described in the disclosure. Unless a specific isoformor subset of isoforms is indicated, the term ERG includes ERG1, ERG2,ERG3, ERG4, ERG5, ERG6, ERG7, ERG8, ERG9, EPC1, EPC2, and the truncatedERG transcripts that result from activation of the prostatecancer-specific promoter described herein. The phrasing “ERG, but not”one or more specifically mentioned ERG isoforms may be used inembodiments in which several different, but not all, of the ERG isoformsare contemplated. The cDNA sequence of the ERG1 gene is published inGenBank under the accession number M21535. The cDNA sequence of the ERG2gene is published in GenBank under the accession number M17254. The term“ERG8” refers to the isoform described, e.g., by SEQ ID NO: 1, SEQ IDNO: 30, and SEQ ID NO: 46. The exon usage of ERG isoforms 1-9 ispresented in Owczarek et al., GENE 324:65-77 (2004). When the contextdoes not dearly exclude it, ERG also refers to the various ERGpolypeptides encoded by the different isoforms. Further, althoughitalics are generally used to refer to nucleic acids, the use of italicsis not to be construed as excluding the encoded polypeptide.

To “destabilize” one or more transcripts means to cause degradation ofthat/those transcript(s) such that expression of the encodedpolypeptide(s) is inhibited or knocked-down. Silent interfering RNA(siRNA), small hairpin RNA (shRNA) (for example, as described byPaddison et al., GENES DEV 16(8):948-58 (2002), antisense molecules,ribozymes, and combinations of these approaches can be used in methodsof destabilizing a transcript(s).

A “moderate risk” prostate cancer is cancer in which the patient has,for example, no PSA recurrence, a Gleason score of 6-7, T2a-T3b stage,no seminal vesicle invasion, and well- or moderately-differentiatedtumor.

A “high risk” prostate cancer is cancer in which the patient as forexample, PSA recurrence, a Gleason score of 8-9, T3c stage, seminalvesicle invasion, and poor tumor differentiation.

The term “altered expression” refers both to qualitative differences(i.e., that gene or protein expression is detectable versusundetectable) and to quantitative differences (i.e., differences inmeasured levels of gene or protein expression).

The term “isolated” refers to a molecule that is substantially free ofits natural environment. Any amount of that molecule elevated over thenaturally occurring levels due to any manipulation, e.g., overexpression, partial purification, etc., is encompassed with thedefinition. With regard to partially purified compositions only, theterm refers to an isolated compound that is at least 50-70%, 70-90%,90-95% (w/w), or more pure.

The phrase “substantially identical,” or “substantially as set out,”means that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 95%,97, 98, or 99% identical to a given sequence. By way of example, suchsequences may be allelic variants, sequences derived from variousspecies, or they may be derived from the given sequence by truncation,deletion, amino acid substitution, or addition. For polypeptides, thelength of comparison sequences will generally be at least 20, 30, 50,100 or more amino acids. For nucleic acids, the length of comparisonsequences will generally be at least 50, 100, 150, 300, or morenucleotides. Percent identity between two sequences is determined bystandard alignment algorithms such as, for example, Basic LocalAlignment Tool (BLAST) described in Altschul et al., J MOL BIOL215:403-410 (1990), the algorithm of Needleman et al., J MOL BIOL48:444-453 (1970), or the algorithm of Meyers et al., COMPUT APPL BIOSCI411-17 (1988).

“Protein” is used interchangeably with the terms “peptide” and“polypeptide” and refers to any chain of amino acids, regardless oflength or posttranslational modification (e.g., glycosylation orphosphorylation), or source (e.g., species).

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “DNA”are used interchangeably herein and refer to deoxyribonucleic acid(DNA), and, where appropriate, ribonucleic acid (RNA). The term shouldalso be understood to include nucleotide analogs, and single or doublestranded polynucleotides. Examples of polynucleotides include, but arenot limited to, plasmid DNA or fragments thereof, viral DNA or RNA,anti-sense RNA, etc. The term “plasmid DNA” refers to double strandedDNA that is circular.

As used herein the term “hybridization under defined conditions,” or“hybridizing under defined conditions,” is intended to describeconditions for hybridization and washes under which nucleotide sequencesthat are significantly identical or homologous to each other remainbound to each other. The conditions are such that sequences, which areat least about six and more preferably at least about 20, 30, 40, 50,100, 150, 300, or more nucleotides long and at least about 70%, morepreferably at least about 80%, even more preferably at least about85-90% identical, remain bound to each other. The percent identity canbe determined as described in Altschul et al., NUCLEIC A CIDS RES25:3389-3402 (1997). Appropriate hybridization conditions can beselected by those skilled in the ad with minimal experimentation asexemplified in Ausubel et al., CURRENT PROTOCOLS IN MOLEC BIOL, JohnWiley & Sons (2004). Additionally, stringent conditions are described inSambrook at al. MOLEC CLONING: A LABORATORY MANUAL, 3^(rd) ed., ColdSpring Harbor Laboratory Press (2001).

A nonlimiting example of defined conditions of low stringency is asfollows: Filters containing DNA are pretreated for six hours at 40° C.in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5),5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA. Hybridizations are carried out in the same solution with thefollowing modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mlsalmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm³²P-labeled probe is used. Filters are incubated in the hybridizationmixture for 18-20 hours at 40° C., and then washed for 1.5 hours at 55°C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA,and 0.1% SDS. The wash solution is replaced with fresh solution andincubated an additional 1.5 hours at 60° C. Filters are blotted dry andexposed for autoradiography. Other conditions of low stringency wellknown in the art may be used (e.g., as employed for cross-specieshybridizations).

A non-limiting example of defined conditions of high stringency is asfollows: Prehybridization of filters containing DNA is carried out for 8hours to overnight at 65° C. in buffer composed of 0×SSC, 50 mM Tris-HCl(pH 7.5), 1 mM EDTA. 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C. in the prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Filters are washed for1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01%Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C.for 45 minutes. Another non-limiting example of defined conditions ofhigh stringency is as follows: Prehybridization of filters containingDNA is carried out for eight hours to overnight at 65° C. in buffercomposed of 6×SSC. 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 12 hours at 65° C. in the prehybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Filters are washed for 1 hour at 37° C. in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This isfollowed by a wash in 0.1×SSC at 50° C. for 45 minutes. Other conditionsof high stringency well known in the art may be used. An oligonucleotidehybridizes specifically to a target sequence under high stringencyconditions.

The term “primer” or “oligonucleotide primer” means an oligonucleotidecapable of binding to a region of a target nucleic acid, or itscomplement, and promoting nucleic acid amplification of the targetnucleic acid. Generally, a primer will have a free 3′ end that can beextended by a nucleic acid polymerase. Primers also generally include abase sequence capable of hybridizing via complementary base interactionseither directly with at least one strand of the target nucleic acid orwith a strand that is complementary to the target sequence. A primer maycomprise target-specific sequences and optionally other sequences thatare non-complementary to the target sequence. These non-complementarysequences may comprise, for example, a promoter sequence or arestriction endonuclease recognition site.

The term “solid support” means a material that is essentially insolubleunder the solvent and temperature conditions of the assay method,comprising free chemical groups available for joining an oligonucleotideor nucleic acid. Preferably, the solid support is covalently coupled toan oligonucleotide designed to directly or indirectly bind a targetnucleic acid. When the target nucleic acid is an mRNA, theoligonucleotide attached to the solid support is preferably a poly-Tsequence. A preferred solid support is a particle, such as a micron- orsubmicron-sized bead or sphere. A variety of solid support materials arecontemplated, such as, for example, silica, polyacrylate,polyacrylamide, a metal, polystyrene, latex, nitrocellulose,polypropylene, nylon or combinations thereof. In some embodiments, thesolid support is capable of being attracted to a location by means of amagnetic field, such as a solid support having a magnetite core.

The term “detecting” or “detection” means any of a variety of methodsknown in the art for determining the presence of a nucleic acid or aprotein. For example, hybridizing a labeled probe to a portion of anucleic acid is one way to detect that nucleic acid. Binding an antibodythat is either directly or indirectly labeled to a protein of interestis an example of a method of detecting that protein. Methods forlabeling nucleic acids and antibodies (as well as other proteins) arewell known in the art. Labels can be either detectable or functionallabels, and include radiolabels (e.g., ¹³¹I, ¹²⁵I, ³⁵S, and ⁹⁹Tc),enzymatic labels (e.g., horseradish peroxidase or alkaline phosphatase),chemiluminescent labels, and other chemical moieties (e.g., biotin). Alabeled probe is an oligonucleotide that specifically binds to anothersequence and contains a detectable group which may be, for example, afluorescent moiety, a chemiluminescent moiety (such as an acridiniumester (AE) moiety that can be detected chemiluminescently underappropriate conditions (as described in U.S. Pat. No. 5,283,174)), aradioisotope, biotin, avidin, enzyme, enzyme substrate, or otherreactive group. Other well known detection techniques include, forexample, gel filtration, gel electrophoresis and visualization of theamplicons by, for example, staining with ethidium bromide, and HighPerformance Liquid Chromatography (HPLC). Antibody-based detectionmethods include ELISA, western blotting, radioimmunoassay (RIA),immunohistochemistry, and other techniques that are well known in theart. As used throughout the specification, the term “detecting” or“detection” includes either qualitative or quantitative detection.

The term “treatment” is used interchangeably herein with the term“therapeutic method” and refers to both therapeutic treatment andprophylactic/preventative measures. Those in need of treatment mayinclude individuals already having a particular medical disorder as wellas those who may ultimately acquire the disorder.

The term “effective dose,” or “effective amount,” refers to that amountof the compound that results in amelioration of symptoms in a patient ora desired biological outcome, e.g., inhibition of cell proliferation.The effective amount can be determined as described in the subsequentsections.

The term “modulatory compound” is used interchangeably with the term“therapeutic” and as used herein means any compound capable of“modulating” either prostate cancer-specific gene expression at thetranscriptional, translational, or post-translational levels ormodulating the biological activity of a prostate cancer-specificpolypeptide. The term “modulate” and its cognates refer to thecapability of a compound acting as either an agonist or an antagonist ofa certain reaction or activity. The term modulate, therefore,encompasses the terms “activate” and “inhibit.” The term “activate,” forexample, refers to an increase in the expression of the prostatecancer-specific gene or activity of a prostate cancer-specificpolypeptide in the presence of a modulatory compound, relative to theactivity of the gene or the polypeptide in the absence of the samecompound. The increase in the expression level or the activity ispreferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or higher. Analogously, the term “inhibit” refers to a decrease in theexpression of the prostate cancer-specific gene or activity of aprostate cancer-specific polypeptide in the presence of a modulatorycompound, relative to the activity of the gene or the polypeptide in theabsence of the same compound. The decrease in the expression level orthe activity is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or higher. The expression level of the prostatecancer-specific gene or activity of a prostate cancer-specificpolypeptide can be measured as described herein or by techniquesgenerally known in the art.

“Antibody” refers to an immunogiobulin or fragment thereof, andencompasses any polypeptide comprising an antigen-binding fragment or anantigen-binding domain. The term includes but is not limited topolyclonal, monoclonal, monospecific, polyspecific, humanized, human,single-chain, chimeric, synthetic, recombinant, hybrid, mutated,grafted, and in vitro generated antibodies. Unless preceded by the word“intact”, the term “antibody” includes antibody fragments such as Fab,F(ab′)₂), Fv, scFv, dAb, and other antibody fragments that retainantigen-binding function. Unless otherwise specified, an antibody is notnecessarily from any particular source, nor is it produced by anyparticular method.

The terms “specific interaction,” “specific binding,” or the like, meanthat two molecules form a complex that is relatively stable underphysiologic conditions. The term is also applicable where, e.g., anantigen-binding domain is specific for a particular epitope, which iscarried by a number of antigens, in which case the specific bindingmember carrying the antigen-binding domain will be able to bind to thevarious antigens carrying the epitope. Specific binding is characterizedby a high affinity and a low to moderate capacity. Nonspecific bindingusually has a low affinity with a moderate to high capacity. Typically,the binding is considered specific when the affinity constant K_(a) ishigher than 10⁶M⁻¹, more preferably higher than 10⁷M⁻¹, and mostpreferably 10⁸ M⁻¹. If necessary, non-specific binding can be reducedwithout substantially affecting specific binding by varying the bindingconditions. Such conditions are known in the art, and a skilled artisanusing routine techniques can select appropriate conditions. Theconditions are usually defined in terms of concentration of antibodies,ionic strength of the solution, temperature, time allowed for binding,concentration of non-related molecules (e.g., serum albumin, milkcasein), etc.

Prostate Cancer-Specific ERG Nucleic Acids

The disclosure describes prostate cancer-specific ERG isoform nucleicacids, in particular, ERG8, EPC1, EPC2, and a prostate cancer-specificpromoter located within exon 9 of the ERG gene. In the case of ERG8, apartial-length splice variant of the ERG gene has been described(Owczarek et al. (2004)), but the complete ERG8 nucleotide sequence andERG8 overexpression in the context of prostate cancer was not previouslyknown. ERG8 is herein reported to comprise 2441 nucleotides. It isoverexpressed in 60-75% of prostate tumors, thus provides a target forthe detection, prognosis, and treatment of prostate cancer.

The protein encoded by ERG8 lacks the DNA binding domain found in ERG-11and ERG2 but retains the entire protein-protein interaction domain. Theexpression of ERG8, therefore, likely results in the functionalnullification of protein interaction partners of ERG1 and ERG2,resulting in a dominant negative effect.

The disclosure also shows that fusions occur between ERG8 and TMPRSS2.An example of a TMPRSS2-ERG8 fusion transcript is:

(SEQ ID NO: 1) TAGGCGCGAG CTAAGCAGGA GGCGGAGGCG GAGGCGGAGG GCGAGGGGCG 50

 100 ACCAGTCGTT CTTTGAGTGT GCCTACGGAA CGCCACACCT GGCTAAGACA  150 GAG

ACCG CGTCCTCCTC CAGCGACTAT GGACAGACTT CCAAGATGAG  200CCCACGCGTC CCTCAGCAGG ATTGGCTGTC TCAACCCCCA GCCAGGGTCA  250

 300 CCTGATGAAT GCAGTGTGGC CAAAGGCGGG AAGATGGTGG GCAGCCCAGA  350CACCGTTGGG ATGRACTACG GCAGCTACAT GGAGGAGAAG CACATGCCAC  400

 450 ACGCTATGGA GTACAGACCA TGTGCGGCAG TGGCTGGAGT GGGCGGTGAA  500AGAATATGGC CTTCCAGACG TCAACATCTT GTTATTCCAG AACATCGATG  550GGAAGGAACT GTGCAAGATG ACCAAGGACG ACTTCCAGAG GCTCACCCCC  600

 650 TCCTCTTCCA CATTTGACTT CAGATGATGT TGATAAAGCC TTACAAAACT  700

 750 CCAAATACTT CAGTATATCC TGAAGCTACG CAAAGAATTA CAACTAGGCC  800

 850 GTCCTGCTGA GATCCGTGCC CTAAGTCACG TGATACAAAG AGAGCTGATC  900CCGGAGCTGA AGCCAGTCCC AGACAGTCTT ATTCTGCCTC TGTTGATTTG  950GAGACTAAAT CCACTCAAAC CATTTCATTC AAAGACCACA CTAAAGGAAT 1000TAAGAGCAGA TTAGCCCTTT AACTAGCTTT TCAGAAAGAC AGATGGGCAA 1050AGAAGGCATC CTGGATGCCT GGCAGTTAGG AATAGGCCGA CTTTTGAACT 1100AACAGAAGGA TCTGTCCCTC CTCGGGGGAA GAGCACAAAA CAAGGACACT 1150CCCCAGATTC ACAGTGAC.The TMPRSS2-derived sequence at nucleotides 1-75 is shown in bold font.Exon junctions are shown in grey boxes. The initiation codon and stopcodon are shown in bold italics. The unique 3′ sequence at nucleotides803-1168 is also shown in bold font. The amino acid sequence of ERG8 is:

(SEQ ID NO: 2) MTASSSSDYG QTSKMSPRVP QQDWLSQPPA RVTIKMENCP SQVNGSRNSP 50DECSVAKGGK MVGSPDTVGM NYGSYMEEKH MPPPNMTTNE RRVIVPADPT 100LWSTDHVRQW LEWAVKEYGL PDVNILLFQN IDGKELCKMT KDDFQRLTPS 150YNADILLSHL NYLRETPLPH LTSDDVDKAL QNSPRLMHAR NTGGAAFIFP 200NTSVYPEATQ RITTRPGTKT PLCDLFIERH PRCPAEIRAL SHVIQRELIP 250ELKPVPDSLI LPLLIWRLNP LKPFHSKTTL KELRAD.The unique carboxy terminus of ERG8 is shown in bold font.

The invention provides an isolated double-stranded nucleic acid moleculecomprising a nucleic acid molecule with the polynucleotide sequence SEQID NO: 1, SEQ ID NO: 30, or SEQ ID NO: 46, or the complement of any ofthese. The entire nucleotide sequence of the ERG8/TMPRSS fusion moleculeis:

(SEQ ID NO: 30) TAGGCGCGAG CTAAGCAGGA GGCGGAGGCG GAGGCGGAGG GCGAGGGGCG 50 GGGAGCGCCG CCTGGAGCGC GGCAGGAAGC CTTATCAGTT GTGAGTGAGG  100ACCAGTCGTT GTTTGAGTGT GCCTACGGAA CGCCACACCT GGCTAAGACA  150 GAG

ACCG CGTCCTCCTC CAGCGACTAT GGACAGACTT CCAAGATGAG  200CCCACGCGTC CCTCAGCAGG ATTGGCTGTC TCAACCCCCA GCCAGGGTCA  250CCATCAAAAT GGAATGTAAC CCTAGCCAGG TGAATGGCTC AAGGAACTCT  300CCTGATGAAT GCAGTGTGGC CAAAGGCGGG AAGATGGTGG GCAGCCCAGA  350CACCGTTGGG ATGAACTACG GCAGCTACAT GGAGGAGAAG CACATGCCAC  400CCCCAAACAT GACCACGAAC GAGCGCAGAG TTATCGTGCC AGCAGATCCT  450ACGCTATGGA GTACAGACCA TGTGCGGCAG TGGCTGGAGT GGGCGGTGAA  500AGAATATGGC CTTCCAGACG TCAACATCTT GTTATTCCAG AACATCGATG  550GGAAGGAACT GTGCAAGATG ACCAAGGACG ACTTCCAGAG GCTCACCCCC  600AGCTACAACG CCGACATCCT TCTCTCACAT CTCCACTACC TCAGAGAGAC  650TCCTCTTCCA CATTTCACTT CAGATGATGT TGATAAAGCC TTACAAAACT  700CTCCACGGTT AATGCATGCT AGAAACACAG GGOGTGCAGC TTTTATTTTC  750CCAAATACTT CAGTATATCC TGAAGCTACG CAAAGAATTA CAACTAGGCC  800AGGTACGAAA ACACCCCTGT GTGATCTCTT CATTGAGAGA CATCCCAGAT  850GTCCTGCTGA GATCCGTGCC CTAAGTCACG TGATACAAAG AGAGCTGATC  900CCGGAGCTGA AGCCAGTCCC AGACAGTCTT ATTCTGCCTC TGTTGATTTG  950GAGACTAAAT CCACTCAAAC CATTTCATTC AAAGACCACA CTAAAGGAAT 1000 TAAGAGCAGA T

CCCTTT AACTAGCTTT TCAGAAAGAC AGATGGGCAA 1050AGAAGGCATC CTGGATGCCT GGCAGTTAGG AATAGGCCGA CTTTTGAACT 1100AACAGAAGGA TCTGTCCCTC CTCGGGGGAA GAGCACAAAA CAAGGAGACT 1150CCCCAGATTC ACAGTGACCG ATTATCAGTA TGTCACAAGA AGCCAGTCTT 1300GCAGAGCAGA AGCATGCAAC CAGTAGTATT TACATCTGAA TCTTACTGCC 1250

1300

1350 CTGGGAATAT ATAGATGAAC CAGGCTTCAG TAAGOTTCQT GTCTTCAGAA 1400AGTTTACTTC TTCATTCAGC TTGGTTTGTT CATTTGCTGA GTGCCTCCTC 1450TGTGCCAGCC ACGGATGGTA TGATGGTGAA CAAACCGAAA TGTTTTGCCT 1500CCAGTTCTAG ATGTTTCAGT AGAGTGACCT AGAGCCAGAG AGACACATAT 1550GTACACATAA ATGTTTTCCC TAATGTGATA GATTTTATGG TAGAGGAACC 1600ACTTCTAGCA ATACAGGGCG TAGGAGCAGG GGTGGGGAGG AACTCAATCC 1650CCCATGAAAG GCATAAAGTT GCTTTCCAGA GGAATGGCCA CATGGCAAAG 1700GGGAATTAGA TGTTTGCCAG ACGAATAATG AGCAGGGAGA GAGGGCATTT 1750CCCAGAAGGG TATAGCTTGC CTTTAGCATT TGTCGTCTCC CTGGGACTTA 1800CATCAGCCCG ATAAGCTAGG TATCATTGTA CCAGCCTCAC AGCTGATGAC 1850ATTGTGTTCA GGGTGGTGGG ATGGTTTCTC CATATTCATA CATGCTTCCA 1900GAATTCATGT TAAACTCTAT CACATATCCG GAATACACAA GTCTCAGTTC 1950GAACTGGTTC AAGATCTAGG CTTGGCAACT ACTCTTTCTT TCTAATGAGA 2000AAGACTGGGG GCCCAGGGAG CTAAAGAGAA TGAATGAGGA AGCTTCTCAG 2050GCTGTTCAAA TACTGACACT GCCCTGGTTA CTGCCTAGTG ACTTCAGGCT 2100GGCAATTTTC TCTTCTCTAA CGTCAGAGAA AAAGTTTACT GTCTTGCTCC 2150TGGGAAGCAT GATGGAAAGG CTTAGCAGCT AAGGGGTACT AAGAGGTAGT 2200AAGTCATCTC TGTCATGTAA AAGATTTCAC AGGCCATTGA AACATGGGCA 2250AGACCCAGTG CCTAGAGTCT GCAAGATTGGT CCTAAAGAC ATCCACCACG 2300TGTATTGCGA GTGGAAAATA GAAATTCATG TTTGACTCAA GCTTTAGAGA 2350TTTTGTAATT CTGTGAGCAT TTAAAAAATA TTTCCATATA AACTAAAAAA 2400ATAAAAACTA TTTCCAAAAA AAAAAAAAAA AAAAACTCGA G. 2441SEQ ID NO: 1 is identical to nucleotides 1-1168 of SEQ ID NO: 30. TheTMPRSS2-derived sequence is shown in bold font and the initiation codonand stop codon are shown in bold italics, also as shown in SEQ ID NO: 1.The shaded region, SEQ D NO: 48, identities an adenosine-rich areacorresponding to nucleotides 1300-1310 of SEQ ID NO: 30. It aligns withthe 3′ end of AY204742, as shown below.

(SEQ ID NO: 48) AAAAATAAACA (SEQ ID NO: 49) AAAAAAAAAAAThe two bolded nucleotides, T1305 and C1309 of SEQ ID NO: 30, bothcorrespond to adenosines in the AY204742 sequence, suggesting thatAY204742 may previously have been erroneously considered to possess a ofpolyA tail at this site.

Nucleotides 802-2415 comprise a region specific to ERG8, This regionencodes the unique carboxy terminus and also comprises a non-coding 3′region, as shown below:

(SEQ ID NO: 46) GTACGAAAAC ACCCCTGTGT GATCTCTTCA TTGAGAGACA TCCCAGATGT50 CCTGCTGAGA TCCGTGCCCT AAGTCACGTG ATACAAAGAG AGCTGATCCC 100GGAGCTGAAG CCAGTCCCAG ACAGTCTTAT TCTGCCTCTG TTGATTTGGA 150GACTAAATCC ACTCAAACCA TTTCATTCAA AGACCACACT AAAGGAATTA 200AGAGCAGATT AGCCCTTTAA CTAGCTTTTC AGAAAGACAG ATGGGCAAAG 250AAGGCATCCT GGATGCCTGG CAGTTAGGAA TAGGCCGACT TTTGAACTAA 300CAGAAGGATC TGTCCCTCCT CGGGGGAAGA GCACAAAACA AGGACACTCC 350CCAGATTCAC AGTGACCGAT TATCAGTATG TCACAAGAAG CCAGTCTTGC 400AGAGCAGAAG CATGCAACCA GTAGTATTTA CATCTGAATC TTACTGCCTG 450TCCTCCAAAT GATTTAATTA GGTAATAAAT TTACATGCCA TTCATGCAAA 500AATAAACATC TATCAAGTGC CCATTAGTGC CAAGCGTGGT GTTAGACTCT 550GGGAATATAT AGATGAACCA GGCTTCAGTA AGCTTCCTGT CTTCAGAAAG 600TTTACTTCTT CATTCAGCTT GGTTTGTTCA TTTGCTGAGT GCCTCCTCTG 650TGCCAGCCAC GGATGGTATG ATGGTGAACA AACCGAAATG TTTTGCCTCC 700AGTTCTAGAT GTTTCAGTAG AGTGACCTAG AGCCAGAGAG ACACATATGT 750ACACATAAAT GTTTTCCCTA ATGTGATAGA TTTTATGGTA GAGGAACCAC 800TTCTAGCAAT ACAGGGCGTA GGAGCAGGGG TGGGGAGGAA CTCAATCCCC 850CATGAAAGGC ATAAAGTTGC TTTCCAGAGG AATGGCCACA TGGCAAAGGG 900GAATTAGATG TTTGCCAGAC GAATAATGAG CAGGGAGAGA GGGCATTTCC 950CAGAAGGGTA TAGCTTGCCT TTAGCATTTG TCCTCTCCCT GGGACTTACA 1000TCAGCCCGAT AAGCTAGGTA TCATTGTACC AGCCTCACAG CTGATGACAT 1050TGTGTTCAGG GTGGTGGGAT GGTTTCTCCA TATTCATACA TGCTTCCAGA 1100ATTCATGTTA AACTCTATCA CATATCCGGA ATACACAAGT CTCAGTTCGA 1150ACTGGTTCAA GATCTAGGCT TGGCAACTAC TCTTTCTTTC TAATGAGAAA 1200GACTGGGGGC CCAGGGAGCT AAAGAGAATG AATGAGGAAG CTTCTCAGGC 1250TGTTCAAATA CTGACACTGC CCTGGTTACT GCCTAGTGAC TTCAGGCTGG 1300CAATTTTCTC TTCTCTAACG TCAGAGAAAA AGTTTACTGT CTTGCTCCTG 1350GGAAGCATGA TGGAAAGGCT TAGCAGCTAA GGGGTACTAA GAGGTAGTAA 1400GTCATCTCTG TCATGTAAAA GATTTCACAG GCCATTGAAA CATGGGCAAG 1450ACCCAGTGCC TAGAGTCTGC AAGATTGGTC CTAAAGACAT CCACCACGTG 1500TATTGCGAGT GGAAAATAGA AATTCATGTT TGACTCAAGC TTTAGAGATT 1550TTGTAATTCT GTGAGCATTT AAAAAATATT TCCATATAAA CTAAAAAAAT 1600AAAAACTATT TCC. 1613The ERG8 specific carboxyl terminus of the encoded polypeptide is:

(SEQ ID NO: 47) GTKTPLCDLF IERHPRCPAE IRALSHVIQR ELIPELKPVPDSLILPLLIW RLNPLKPFHS KTTLKELRAD.

EPC1 is an ERG isoform that is selectively expressed in cancerousprostate cells. The nucleic acid sequence of EPC1 is:

(SEQ ID NO: 3) GCAGGAGGCG GAGGCGGAGG CGGAGGGCGA GGGGCGGGGA GCGCCGCCTG 50

 100 GAGTGTGCCT ACGGAACGCC ACACCTGGCT AAGACAGAG

ACCGCGTC  150 CTCCTCCAGC GACTATGGAC AGACTTCCAA GATGAGCCCA CGCGTCCCTC 200 AGCAGGATTG GCTGTCTCAA CCCCCAGCCA GGGTCACCAT CAAAATGGAA  250

 300 TGTGGCCAAA GGCGGGAAGA TGGTGGGCAG CCCAGACACC GTTGGGATGA  350ACTACGGCAG CTACATGGAG GAGAAGCACA TGCCACCCCC AAACATGACC  400

 450 AGACCATGTG CGGCAGTGGC TGGAGTGGGC GGTGAAAGAA TATGGCCTTC  500CACACGTCAA CATCTTGTTA TTCCAGAACA TCGATGGGAA GGAACTGTGC  550AAGATGACCA AGGACGACTT CCAGAGGCTC ACCCCCAGCT ACAACGCCGA  600

 650 TGACTTCAGA TGATGTTGAT AAAGCCTTAC AAAACTCTCC ACGGTTAATG  700

 750

 800

AACAACAG AACCAGTGCC AGAAAGCAGC CTTCCCTTAC ATGGGCACTT  850CTGCCAAGCA TATGAGTTCA TTGCCTTGAA GATCAAAGTC AAAGAGAAAT  900GGAGAGGGTG TTGAAATGAT CAGCGAAAAT TAAATGTAAA ATATATTCTT  950ATTGGAAGTC TGATGCTCTA TTATCAATAA AGGACACATA GCAAAGATAA 1000AAAAAAAAAA AAAAAAAAAA.In the sequence, the TMPRSS2-derived sequence is shown in bold font.Exon junctions are shown in grey boxes. The initiation codon and stopcodon are shown in bold italics. The 3′ end of the EPC1 transcript isdistinct from all known ERG isoforms. This unique sequence is shown inbold font. The amino acid sequence of EPC1 is:

(SEQ ID NO: 4) MTASSSSDYG QTSKMSPRVP QQDWLSQPPA RVTIKMECNP SQVNGSRNSP 50DECSVAVKKG MVGSPDTVGM MYGSYMEEKH MPPPNMTTNE RRVIVPADPT 100LWSTDHVRQW LEWAVKEYGL PDVNILLFQN IDGKELCKMT KDDFQRLTPS 150YNADILLSHL HYLRETPLPH LTSDDVDKAL QNSPRLMHAR NTGGAAFIFP 200NTSVYPEATQ RITTRPVSYR.EPC1 comprises additional nucleotides at its 3′ end that encode fourunique amino acids at the carboxy terminus of the EPC1 protein. Thesefour unique amino acids are underlined in SEQ ID NO: 4. Because EPC1,like ERG8, lacks the coding sequences for the DNA-binding domain, it mayalso have a dominant negative effect.

EPC2 is also selectively expressed in cancerous prostate cells. Thenucleic acid sequence of EPC2 is:

(SEQ ID NO: 5) ACATCTTGTT ATTCCAGAAC ATCGATGGGA AGGAACTGTG CAAG

ACC  50 AAGGACGACT TCCAGAGGCT CACCCCCAGC TACAACGCCG ACATCCTTCT  100

150 TACATGGCTG TGGCTATGGT TCTTATCACC CGAGCTTCAG AGGGTTCAAC 200CAGGTGTGTC GACAGCATCC TCCTGCCCTC GCCCAGTTCC CACTCGGGAT 250CCGAGGCAGC CACATCCTTG GGTCCTGCGA CCAAGAAGAT GGAATGTCAA 300AGGGGAAAGG AAGCGTTAAC TGGTCACACA TTAGTTAAGT CTCCATGATA 350CCCCGAATCA AAATAGAATC ATTAAGGCTT CTCTTTCGTA CGAATTAGGC 400GGATTATTCT CCCTAAAGCT ACATGAAGCC CCACTTTATA TTCTAACCTC 480AGCACAGAAC AACCGAAGTT TTCACTTTCT ATCATGTGAT TCGGCTTAAC  500CTGACAGAAA OCGATGGCAT CTTGGCATCA ATCCAGAATG TTTGCTCCAT 550GCTTTAATTT CTACAACGTC CAGCATGGTG AGAAGGAAGT AGTGTGACAG 500ACAGTGAGCT GGATAAATTC TCCTCCATTG CTTTGCCTGG CATCCCAACC 650ACTTCTTCCC TGAATTAAAG ACGGGCCCCC ATGTAGGTTT TAACATGCTA 700ACAACTAGCA GGTTGCTGGA AATAGTTATA AGCTTCCCAT GATGTTAGTG 750TGGGACTGGG GGAACCGTTT CTTTCTTTCT TTTTCTTTCT TTTTTTTTTT 800 TTTTTTT.The initiation codon and stop codon are shown in bold italics. An exonjunction is shown in the grey box. The unique 3′ sequence is shown inbold font. The amino acid sequence of EPC2 is:

(SEQ ID NO: 6) MTKDDFQRLT PSYNADILLS HLHYLRESKL PLPPRIDGCG YGSYDPSFRG 50 FNQVCRQHPP ALAQFPLGIR GSHMLGSCDQ EDGMSKGKGS VNWSHISThe unique carboxy terminus of EPC2 is shown in bold font in SEQ ID NO:6.

The disclosure also describes the activation of a promoter in prostatecancer cells. Activation of this promoter produces transcripts codingfor ERG isoforms lacking the n-terminal protein-protein interactiondomain of wild type ERG. Therefore, expression products of this promotersequence in prostate cancer cells appear to act as dominant negative orgain-of-function molecules. The promoter is located within the followingsequence from exon 9 of the ERG gene:

In the sequence, the most 3′ transcription start site is bolded andshown in a grey box. A sequence comprising at least nucleotides 521 to650 of SEQ ID NO: 7 retain promoter activity.

Diagnostic Compositions and Methods

The ERG isoform nucleic acids, the polypeptides they encode, andantibodies to those polypeptides can be employed in various diagnosticand prognostic applications for prostate cancer because ERG8, EPC1,EPC2, and the transcripts from the prostate cancer-specific promoter areeach associated with prostate cancer.

Accordingly, the disclosure provides methods for detecting prostatecancer in a biological sample, comprising combining the biologicalsample with at least a first and a second oligonucleotide primer underhybridizing conditions, wherein the first oligonucleotide primercontains a sequence that hybridizes to a first sequence in a targetsequence from ERG8, EPC1, EPC2, or the transcripts from the prostatecancer-specific promoter and the second oligonucleotide primer containsa sequence that hybridizes to a second sequence in a nucleic acid strandcomplementary to the target sequence, wherein the first sequence doesnot overlap with the second sequence; amplifying a plurality ofamplification products when the target sequence is present in thebiological sample by adding at least one polymerase activity to thebiological sample containing the first and second oligonucleotideprimers; immobilizing the plurality of amplification products on a solidsupport; combining an oligonucleotide probe with the immobilizedplurality of amplification products to thereby permit the probe tohybridize to at least one immobilized amplification product; anddetecting whether a signal results from hybridization between theoligonucleotide probe and at least one amplification product, whereindetection of the signal indicates the expression of ERG8, EPC1, EPC2, orthe transcripts from the prostate cancer-specific promoter and thepresence of prostate cancer in the biological sample. Detecting a signalresulting from hybridization between the oligonucleotide probe and theat least one amplification product can be used to diagnose or prognoseprostate cancer.

In some embodiments in which the ERG isoform is fused to TMPRSS2, thefirst oligonucleotide primer contains a sequence that hybridizes to afirst sequence in a target sequence from TMPRSS2 and the secondoligonucleotide primer contains a sequence that hybridizes to a secondsequence in a nucleic acid strand complementary to a target sequencefrom ERG8, EPC1, EPC2, or the transcripts from the prostatecancer-specific promoter.

Accordingly, the disclosure provides methods for detecting prostatecancer in a biological sample, wherein the target sequence comprises allor part of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 30, or SEQ ID NO: 46. In other embodiments, the target sequencecomprises nucleotides 75 to 1168 of SEQ ID NO: 1, nucleotides 803 to1168 of SEQ ID NO: 1, nucleotides 61 to 1019 of SEQ ID NO: 3,nucleotides 788 to 1019 of SEQ ID NO: 3, a nucleic acid moleculecomprising SEQ ID NO: 5, nucleotides 127 to 807 of SEQ ID NO: 5,nucleotides 942-1046 of SEQ ID NO: 46, nucleotides 1285-1374 of SEQ IDNO: 46, or nucleotides 1451-1541 of SEQ ID NO: 46.

In some embodiments, the oligonucleotide probe(s), rather than theamplification products, may be optionally fixed to a solid support.

In yet other embodiments, the immobilization and subsequent steps areomitted and the plurality of amplification products are detected by sizeseparation followed by staining with a reagent, such as ethidiumbromide, that detects DNA. This embodiment may optionally furthercomprise photographing the stained DNA to preserve the results. In theseembodiments detection of the amplification products can be used todiagnose or prognose prostate cancer as well.

When detecting ERG isoform expression in a biological sample, theoligonucleotide probe, first oligonucleotide primer, and secondoligonucleotide primer, each comprise a nucleic acid sequence that iscapable of hybridizing under defined conditions (for example under highstringency hybridization conditions; such as hybridization for 12 hoursat 65° C. in 6×SSC followed by a wash in 0.1×SSC at 50° C. for 45minutes) to a nucleic acid sequence of an ERG isoform. Thus, theoligonucleotide probe, first oligonucleotide primer, and secondoligonucleotide primer comprises, for example, a nucleic acid sequenceof an ERG isoform, such as SEQ ID NO: 1 (ERG8), SEQ ID NO: 3 (EPC1), SEQID NO: 5 (EPC2), SEQ ID NO: 30 (ERG8), SEQ ID NO: 46 (ERG8), atranscript from the prostate cancer-specific promoter (SEQ ID NO: 7), ora nucleic acid molecule comprising a fragment thereof, or a sequencecomplementary thereto. The oligonucleotide probe, first oligonucleotideprimer, or second oligonucleotide primer can be a fragment comprising atleast about 15, at least about 20, at least about 30, at least about 40,or at least about 50 contiguous nucleotides of a nucleic acid sequenceof ERG8, EPC1, EPC2, or a transcript from the prostate cancer-specificpromoter, or a sequence complementary thereto.

In some embodiments, the methods comprise detecting the expression ofthe ERG8 isoform. In other embodiments, expression of the EPC1 isoformis detected. In yet other embodiments, expression of the EPC2 isoform isdetected. While in some embodiments, transcripts from the prostatecancer-specific promoter are detected. In still other embodiments, themethods comprise detecting the ERG8 and EPC1 isoforms combination, theERG8 and EPC2 isoforms in combination, the EPC1 and EPC2 isoforms incombination, or the combination of the ERG8, EPC1, and EPC2 isoforms. Inother embodiments, the method comprises detecting one or moretranscripts from the prostate cancer-specific promoter either alone orin combination with one or more of ERG8, EPC1, or EPC2. In someembodiments, the methods further comprise detecting other prostatecancer-specific markers, such as ERG1, ERG2, PSA, DD3, AMAR, LTF, NPY,SPOCK, CRISP3, PLA2G7, TMEFF2, F5, SMOC, ACPP, TGM4, MSMB, WIF1, OLFM4,PI15, PDGFD, CHGA, CAV1, RLN1, IGFBP7, BGN, FMOD, AGR2, SERPINA3, AZGP1,FAM3B, CD164, or the presence of a TMPRSS-ERG fusion.

Polypeptides encoded by ERG8, EPC1, or EPC2 can also be detected and/ormeasured in a biological sample. For example, antibodies, optionallylabeled, can be used to detect each polypeptide using well knowntechniques, such as ELISA. The biological sample can be, e.g., prostatetissue, blood, serum, plasma, urine, saliva, or prostatic fluid.

In another aspect, the disclosure provides a method of diagnosing orprognosing prostate cancer, comprising measuring the expression level(e.g. mRNA or polypeptide) of ERG8, EPC1, EPC2 or a transcript from theprostate cancer-specific promoter; and correlation the expression levelof an ERG isoform with the presence of prostate cancer or a higherpredisposition to develop prostate cancer in the subject.

The skilled artisan will understand how to correlate expression levelsor patterns of ERG8, EPC1, EPC2, or a transcript from the prostatecancer-specific promoter with the presence of prostate cancer or ahigher predisposition to develop prostate cancer. For example, theexpression levels can be quantified such that increased or decreasedexpression levels relative to a control sample or other standardizedvalue or numerical range indicate the presence of prostate cancer or ahigher predisposition to develop prostate cancer.

The increased or decreased expression levels may be measured relative tothe expression level of ERG8, EPC1, EPC2, or a transcript from theprostate cancer-specific promoter, or the corresponding polypeptide, innormal, matched tissue, such as benign prostate epithelial cells fromthe same subject. Alternatively, the expression level of ERG8, EPC1,EPC2, or a transcript from the prostate cancer-specific promoter, or thecorresponding polypeptide, may be measured relative to the expression ofthe gene or polypeptide in other noncancerous samples from the subjector in samples obtained from an individual who does not have cancer.Expression of a gene or the corresponding polypeptide may also benormalized by comparing it to the expression of other cancer-specificmarkers. For example, a prostate specific marker, such as PSA orTMPRSS2-ERG, can be used as to control to compare and/or normalizeexpression levels of ERG8, EPC1, EPC2, or a transcript from the prostatecancer-specific promoter, or the corresponding polypeptide.

By way of example, the method of diagnosing or prognosing prostatecancer can comprise measuring the expression levels of the ERG8, EPC1,EPC2, or a transcript from the prostate cancer-specific promoter,isoforms, or any combination thereof, and diagnosing or prognosingprostate cancer, where an increased expression level of ERG8, EPC1, orEPC2 of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, or more, as compared to the control sample indicates the presenceof prostate cancer or a higher predisposition in the subject to developprostate cancer, or indicates the severity or stage of prostate cancer,such as whether the cancer is a high risk or a moderate risk prostatecancer.

The expression levels of ERG8, EPC1, EPC2, or a transcript from theprostate cancer specific promoter (e.g., mRNA or polypeptide expression)can be detected according to the methods described herein or using anyother known detection methods, including, without limitation,immunohistochemistry, Southern blotting, northern blotting, westernblotting, ELISA, and nucleic acid amplification procedures that includebut not limited to PCR, transcription-mediated amplification (TMA),nucleic acid sequence-based amplification (NASBA), self-sustainedsequence replication (3SR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), and loop-mediated isothermalamplification (LAMP).

Nucleic acids are also provided for detecting prostate cancer, and oneor more of these nucleic acids may optionally be provided as part of akit. In some embodiments, the nucleic acid is a nucleic acid probe, suchas the probes described elsewhere in the disclosure, that hybridizes toa prostate cancer-specific transcript. For example, in one embodiment,the probe is capable of hybridizing to the desired sequence under highstringency hybridization conditions, such as hybridization for 12 hoursat 65° C. in 6×SSC followed by a wash in 0.1×SSC at 50° C. for 45minutes. The probe can include SEQ ID NO: 1, SEQ ID NO: 30, or SEQ IDNO: 46 itself, or a fragment of SEQ ID NO: 1, SEQ ID NO: 30, or SEQ IDNO: 46 comprising at least about 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, or 200 contiguous nucleotides of SEQ ID NO: 1, SEQ ID NO: 30,or SEQ ID NO: 46, or a sequence complementary thereto. In oneembodiment, the fragment comprises all or part of nucleotides 75 to 1168of SEQ ID NO: 1. For example, the fragment may comprise nucleotides 801to 1168 of SEQ ID NO: 1, or a nucleic acid molecule comprising at leastabout 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 contiguousnucleotides of nucleotides 801 to 1168 of SEQ ID NO: 1. Also by way ofexample, the fragment may comprise nucleotides 942-1046 of SEQ ID NO:46, nucleotides 1285-1374 of SEQ ID NO: 46, or nucleotides 1451-1541 ofSEQ ID NO: 46.

In some embodiments, the probe selectively hybridizes to the ERG8isoform but does not hybridize to ERG1, ERG2, ERG3, ERG4, ERG5, ERG6,ERG7, ERG9, EPC1, EPC2, a transcript from the prostate cancer-specificpromoter, or TMPRSS2 under defined conditions, including, for example,high stringency hybridization conditions. The length of the probe mayvary depending, for example, on the hybridization conditions and thepercent identify between the target sequence and the probe, and,therefore can be up to about 6, 10, 20, 30, 40, 50, 100, 150, 200, 300,400, or 500 nucleotides long.

In some embodiments, therefore, the disclosure provides an isolatednucleic acid comprising at least about 15 contiguous nucleotides ofnucleotides 801 to 1168 of SEQ ID NO: 1, wherein the nucleic acid iscapable of hybridizing to SEQ ID NO: 1, or the complement thereof, underconditions of high stringency but not to ERG1, ERG2, ERG3, ERG4, ERG5,ERG6, ERG7, ERG9, EPC1, EPC2, a transcript from the prostatecancer-specific promoter, or TMPRSS2. In some embodiments, the nucleicacid is up to about 50 nucleotides long. In other embodiments the probeis capable of hybridizing to the desired sequence under conditions ofhigh stringency comprising hybridization for 12 hours at 65° C. in 6×SSCfollowed by a wash in 0.1×SSC at 50° C. for 45 minutes.

In another embodiment, the probe hybridizes to SEQ ID NO: 3, or to asequence within nucleotides 61 to 1019 or 788 to 1058 of SEQ ID NO: 3(EPC1), or to the complement thereof, under defined hybridizationconditions. For example, in one embodiment, the probe is capable ofhybridizing to the desired sequence under high stringency hybridizationconditions, such as, hybridization for 12 hours at 65° C. in 6×SSCfollowed by a wash in 0.1×SSC at 50° C. for 45 minutes. The probe caninclude SEQ ID NO: 3 itself, or a fragment of SEQ ID NO: 3 comprising atleast about 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200contiguous nucleotides of SEQ ID NO: 3, or a sequence complementarythereto. In one embodiment, the fragment comprises all or part ofnucleotides 61 to 1019 of SEQ ID NO: 3. For example, the fragment maycomprise nucleotides 788 to 1019 of SEQ ID NO: 3, or a nucleic acidmolecule comprising at least about 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, or 200 contiguous nucleotides of nucleotides 788 to 1019 ofSEQ ID NO: 3. In some embodiments, the probe selectively hybridizes toEPC1 but not to ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG7, ERG8, ERG9,EPC2, a transcript from the prostate cancer-specific promoter, orTMPRSS2 under defined conditions, including, for example, highstringency hybridization conditions. The length of the probe may varydepending, for example, on the hybridization conditions and the percentidentify between the target sequence and the probe, and, therefore canbe up to about 0, 10, 20, 30 40, 50, 100, 150, 200, 300 400, or 500nucleotides long.

In some embodiments, therefore, the disclosure provides an isolatednucleic acid comprising at least about 15 contiguous nucleotides ofnucleotides 788 to 1019 of SEQ ID NO: 3, wherein the nucleic acid iscapable of hybridizing to SEQ ID NO: 3, or the complement thereof, underconditions of high stringency but not to ERG1, ERG2, ERG3, ERG4, ERG5,ERG6, ERG7, ERG8, ERG9, EPC2, a transcript from the prostatecancer-specific promoter, or TMPRSS2. In some embodiments, the nucleicacid is up to about 50 nucleotides long. In other embodiments the probeis capable of hybridizing to the desired sequence under conditions ofhigh stringency comprising hybridization for 12 hours at 65° C. in 6×SSCfollowed by a wash in 0.1×SSC at 50° C. for 45 minutes.

In a further embodiment, the probe hybridizes to SEQ ID NO: 5 (EPC2) orto nucleotides 127 to 807 of SEQ ID NO: 5, or to the complement thereof,under defined hybridization conditions. For example, in one embodiment,the probe is capable of hybridizing to the desired sequence under highstringency hybridization conditions, such as, hybridization for 12 hoursat 65° C. in 6×SSC followed by a wash in 0.1×SSC at 50° C. for 45minutes. The probe can include SEQ ID NO: 5 itself, or a fragment of SEQID NO: 5 comprising at least about 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, or 200 contiguous nucleotides of SEQ ID NO: 5 or a sequencecomplementary thereto. In one embodiment, the fragment comprises all orpart of nucleotides 127 to 807 of SEQ ID NO: 5. For example, thefragment may comprise nucleotides 127 to 807 of SEQ ID NO: 5, or anucleic acid molecule comprising at least about 15, 20, 30, 40, 60, 60,70, 80, 90, 100, 150, or 200 contiguous nucleotides of nucleotides 127to 807 of SEQ ID NO: 5. In some embodiments, the probe selectivelyhybridizes to EPC2 but not to ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG7,ERG8, ERG9, EPC1, a transcript from the prostate cancer-specificpromoter, or TMPRSS2 under defined conditions, including, for example,high stringency hybridization conditions. The length of the probe mayvary depending, for example, on the hybridization conditions and thepercent identify between the target sequence and the probe, and,therefore can be up to about 6, 10, 20, 30, 40, 50, 100, 150, 200, 300,400, or 500 nucleotides long.

In some embodiments, therefore, the disclosure provides an isolatednucleic acid, comprising at least about 15 contiguous nucleotides ofnucleotides 127 to 807 of SEQ ID NO: 5, wherein the nucleic acid iscapable of hybridizing to SEQ ID NO: 6, or the complement thereof, underconditions of high stringency but not to ERG1, ERG2, ERG3, ERG4, ERG5,ERG6, ERG7, ERG8, ERG9, EPC2, a transcript from the prostatecancer-specific promoter, or TMPRSS2. In some embodiments, the nucleicacid is up to about 50 nucleotides long. In other embodiments the probeis capable of hybridizing to the desired sequence under conditions ofhigh stringency comprising hybridization for 12 hours at 65° C. in 6×SSCfollowed by a was in 0.1×SSC at 50° C. for 45 minutes.

In some embodiments, therefore, the disclosure provides an isolatednucleic acid comprising at least about 15 contiguous nucleotides ofnucleotides 942-1056, 1285-1374, or 1451-1541 of SEQ ID NO: 46, whereinthe nucleic acid is capable of hybridizing to SEQ ID NO: 46, or thecomplement thereof, under conditions of high stringency but not to ERG1,ERG2, ERG3, ERG4, ERG5, ERG6, ERG7, ERG9, EPC1, EPC2, a transcript fromthe prostate cancer-specific promoter, or TMPRSS2. In some embodiments,the nucleic acid is up to about 50 nucleotides long. In otherembodiments, the probe is capable of hybridizing to the desired sequenceunder conditions of high stringency comprising hybridization for 12hours at 65° C. in 6×SSC followed by a wash in 0.1× SSC at 50° C. for 45minutes.

A nucleic acid probe may be optionally fixed to a solid support.

In other embodiments, the nucleic acid is en oligonucleotide primer. Thedisclosure provides a number of oligonucleotide primers and primerpairs, such as those described in the examples. In some embodiments, anoligonucleotide primer pair comprise a first oligonucleotide primer anda second oligonucleotide primer, where the first oligonucleotide primercontains a sequence that hybridizes to a first sequence in SEQ ID NO: 1,SEQ ID NO: 30, and/or SEQ ID NO: 46 and the second oligonucleotideprimer contains a sequence that hybridizes to a second sequence in anucleic acid strand complementary to SEQ ID NO: 1, SEQ ID NO: 30, and/orSEQ ID NO: 46, wherein the first sequence does not overlap with thesecond sequence. The first and second oligonucleotide primers arecapable of amplifying a target sequence of interest in ERG8. Thus, insome embodiments The primer pairs amplify a target sequence comprisingall or part of nucleotides 75 to 1168 of SEQ ID NO: 1, all or part ofnucleotides 801 to 1168 of SEQ ID NO: 1, all or part of nucleotides942-1046 of SEQ ID NO: 46, all or part of nucleotides 1285-1374 of SEQID NO: 46, or all or part of nucleotides 1451-1541 of SEQ ID NO: 46. Inother embodiments, the target sequence comprises a nucleic acid moleculewithin nucleotides 75 to 1168 of SEQ ID NO: 1, nucleotides 801 to 1168of SEQ ID NO: 1, or nucleotides 942-1046 of SEQ ID NO: 46, nucleotides1285-1374 of SEQ ID NO: 46, or nucleotides 1451-1541 of SEQ ID NO: 46.

In some embodiments, the primer pair amplifies a target sequence thatselectively hybridizes to the ERG8 isoform but does not hybridize toERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG7, ERG9, EPC1, EPC2, a transcriptfrom the prostate cancer-specific promoter, or TMPRSS2 under definedconditions, including, for example, high stringency hybridizationconditions, such as, hybridization for 12 hours at 65° C. in 6×SSCfollowed by a wash in 0.1×SSC at 50° C. for 45 minutes.

In yet other embodiments, an oligonucleotide primer pair comprise afirst oligonucleotide primer and a second oligonucleotide primer, wherethe first oligonucleotide primer contains a sequence that hybridizes toa first sequence in SEQ ID NO: 3 and the second oligonucleotide primercontains a sequence that hybridizes to a second sequence in a nucleicacid strand complementary to SEQ ID NO: 3, wherein the first sequencedoes not overlap with the second sequence. The first and secondoligonucleotide primers are capable of amplifying a target sequence ofinterest in EPC1. Thus, in some embodiments the primer pairs amplify atarget sequence comprising all or part of nucleotides 61 to 1019 of SEQID NO: 3 or all or part of nucleotides 788 to 1019 of SEQ ID NO: 3. Inother embodiments, the target sequence comprises a nucleic acid moleculewithin nucleotides 61 to 1019 of SEQ ID NO: 3 or nucleotides 788 to 1019of SEQ ID NO: 3. In some embodiments, the primer pair amplify a targetsequence that selectively hybridizes to the EPC1 isoform but do nothybridize to ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG7, ERG8, ERG9, EPC2,a transcript from the prostate cancer-specific promoter, or TMPRSS2under defined conditions, including, for example, high stringencyhybridization conditions, such as, hybridization for 12 hours at 65° C.in 6×SSC followed by a wash in 0.1×SSC at 50° C. for 45 minutes.

In still other embodiments, an oligonucleotide primer pair comprise afirst oligonucleotide primer and a second oligonucleotide primer, wherethe first oligonucleotide primer contains a sequence that hybridizes toa first sequence in SEQ ID NO: 5 and the second oligonucleotide primercontains a sequence that hybridizes to a second sequence in a nucleicacid strand complementary to SEQ ID NO: 5, wherein the first sequencedoes not overlap with the second sequence. The first and secondoligonucleotide primers are capable of amplifying a target sequence ofinterest in EPC2. Thus, in some embodiments the primer pairs amplify atarget sequence comprising all or part of SEQ ID NO: 5 or all or part ofnucleotides 127 to 807 of SEQ ID NO: 5. In other embodiments, the targetsequence comprises a nucleic acid molecule within SEQ ID NO: 6 ornucleotides 127 to 807 of SEQ ID NO: 5. In some embodiments, the primerpair amplify a target sequence that selectively hybridizes to the EPC2isoform but do not hybridize to ERG1, ERG2, ERG3, ERG4, ERG5, ERG6,ERG7, ERG8, ERG9, EPC1, a transcript from the prostate cancer-specificpromoter, or TMPRSS2 under defined conditions, including, for example,high stringency hybridization conditions, such as, hybridization for 12hours at t35° C. in 6×SSC followed by a wash in 0.1×SSC at 50° C. for 45minutes.

The oligonucleotide primers and primer pairs can be provided in kitform. In some embodiments, the kits comprise a pair of oligonucleotideprimers that is capable of amplifying a target sequence of interest inERG8, such as those discussed elsewhere it the disclosure, a pair ofoligonucleotide primers that is capable of amplifying a target sequenceof interest in ERG1, such as those discussed elsewhere in thedisclosure, and/or a pair of oligonucleotide primers that is capable ofamplifying a target sequence of interest in EPC2, such as thosediscussed elsewhere in the disclosure. In this and other embodiments, itis not necessary for the oligonucleotide primers to all have differentsequences. For example, it is possible to amplify target sequences thatare specific for each of ERG8, EPC1, EPC2, or a transcript from theprostate cancer-specific promoter, by selecting an oligonucleotideprimer that hybridizes to a nucleotide sequence, or complement thereof,that is unique to ERG8, an oligonucleotide primer that hybridizes to anucleotide sequence, or complement thereof, that is unique to EPC1, anoligonucleotide primer that hybridizes to a nucleotide sequence, orcomplement thereof, that is unique to EPC2, an oligonucleotide primerthat hybridizes to a nucleotide sequence, or complement thereof, that isunique to a transcript from the prostate cancer-specific promoter, andan oligonucleotide primer that hybridizes to a nucleotide sequence, orcomplement thereof, that is shared by ERG8, EPC1, and EPC2. Thus, it ispossible to use only four oligonucleotide primers to selectively amplifytarget sequences in each of ERG8, EPC1, and EPC2. Other combinations ofprimers can be selected to amplify, for example, ERG8 and EPC1, ERG8 andEPC2, EPC1 and EPC2, or one of more of those isoforms in combinationwith a transcript from the prostate cancer-specific promoter.

The disclosure additionally describes diagnostic kits comprising ananti-ERG isoform-specific antibody, for example, an anti-ERG8 antibody,an anti-EPC1 antibody, or anti-EPC2 antibody. In one embodiment, thedisclosure provides an anti-EPC1 antibody that binds an epitopecomprising amino acids 217 to 220 of SEQ ID NO: 4. In anotherembodiment, the antibody is an anti-EPC2 antibody that binds an epitopewithin or comprising amino acids 28 to 97 of SEQ ID NO: 6. In eithercase, the epitope can be a linear epitope or a conformational epitope.In some embodiments, combinations of antibodies can be included in thekit. For example, a kit can comprise an anti-ERG8 and an anti-EPC1antibody, an anti-ERG8 and an anti-EPC2 antibody, an anti-EPC1 and ananti-EPC2 antibody, or an anti-ERG8, anti-EPC1, and an anti-EPC2antibody. The antibodies can be, optionally, detectably labeled. Theantibodies can be used in both diagnostic and prognostic applications,as described for the nucleic acid probes and primers.

The nucleic acids, polypeptides, and antibodies for use in diagnosingand prognosing prostate cancer are generally formulated with apharmaceutically acceptable carrier. When a nucleic acid, polypeptide,or antibody is part of a kit, an agent that reduces or inhibits thegrowth of microorganisms, such as sodium azide, can optionally beincluded in the formulation.

Therapeutic Compositions and Methods

The ERG isoform (e.g., ERG8, EPC1, EPC2, a transcript from the prostatecancer-specific promoter, ERG1, ERG2, or ERG3) nucleic acids, thepolypeptides they encode, and antibodies to those polypeptides can becombined with a suitable pharmaceutical carrier. The resultingpharmaceutical compositions can be used in various applications, such asdiagnostic applications already described, and also in therapeuticapplications. When the application is therapeutic, the compositionscomprise a therapeutically effective amount of the nucleic acid,polypeptide, or antibody and a pharmaceutically acceptable carrier orexcipient. Such a carrier includes, but is not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.

In therapeutic applications, the ERG isoform (e.g., ERG8, EPC1, EPC2, atranscript from the prostate cancer-specific promoter, ERG1, ERG2, orERG3) nucleic acids, polypeptides, compounds used for destabilization,small molecule inhibitors, and antibody compositions will be formulatedand dosed in a fashion consistent with good medical practice, takinginto account the clinical condition of the individual subject, the siteof delivery, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The effectiveamount of ERG isoform (e.g., ERG8, EPC1, EPC2, a transcript from theprostate cancer-specific promoter, ERG1, ERG2, or ERG3) nucleic acids,polypeptides, compounds used for destabilization, small moleculeinhibitors, and antibody compositions for purposes herein is thusdetermined by such considerations.

The disclosure also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions described. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration. In addition, the ERG isoform(e.g., ERG8, EPC1, EPC2, a transcript from the prostate cancer-specificpromoter, ERG1, ERG2, or ERG3) nucleic acids, polypeptides, compoundsused for destabilization, small molecule inhibitors, and antibodycompositions may be employed in conjunction with other therapeuticcompounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10micrograms/kg body weight and in most cases they will be administered inan amount not in excess of about 8 milligrams/kg body weight per day.

In pharmaceutical dosage forms, the disclosed compositions can beadministered in the form of their pharmaceutically acceptable salts, orthey can also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The subjectcompositions are formulated in accordance to the mode of potentialadministration. Administration of the agents can be achieved in variousways, including oral, buccal, nasal, rectal, parenteral,intraperitoneal, intradermal, transdermal, subcutaneous, intravenous,intra-arterial, intracardiac, intraventricular, intracranial,intratracheal, and intrathecal administration, etc., or otherwise byimplantation or inhalation. Thus, the subject compositions can beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, enemas, injections, inhalants and aerosols.Methods and excipients mentioned elsewhere in the disclosure are merelyexemplary and are in no way limiting.

Compositions for oral administration can form solutions, suspensions,tablets, pills, granules, capsules, sustained release formulations, oralrinses, or powders. For oral preparations, the agents, polynucleotides,and polypeptides can be used alone or in combination with appropriateadditives, for example, with conventional additives, such as lactose,mannitol, corn starch, or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch, orgelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives, and flavoring agents.

The ERG isoform (e.g., ERG8, EPC1, EPC2, a transcript from the prostatecancer-specific promoter, ERG1, ERG2, or ERG3) nucleic acids,polypeptides, compounds used for destabilization, small moleculeinhibitors, and antibody compositions can be formulated intopreparations for injection by dissolving, suspending, or emulsifyingthem in an aqueous or nonaqueous solvent, such as vegetable or othersimilar oils, synthetic aliphatic acid glycerides, esters of higheraliphatic acids or propylene glycol; and if desired, with conventionaladditives such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers and preservatives. Other formulationsfor oral or parenteral delivery can also be used, as conventional in theart.

The ERG isoform (e.g., ERG8, EPC1, EPC2, a transcript from the prostatecancer-specific promoter, ERG1, ERG2, or ERG3) nucleic acids,polypeptides, compounds used for destabilization, small moleculeinhibitors, and antibody compositions can also be introduced intotissues or host cells by other routes, such as viral infection,microinjection, or vesicle fusion. For example, expression vectors canbe used to introduce nucleic acid compositions into a cell as describedherein. Further, jet injection can be used for intramuscularadministration (Furth et al., ANAL BIOCHEM 205:365-368 (1992)). The DNAcan be coated onto gold microparticles, and delivered intradermally by aparticle bombardment device, or “gene gun” as described in theliterature (Tang et al., NATURE 356:152-154 (1992)), where goldmicroprojectiles are coated with the DNA, then bombarded into skincells.

In some embodiments, nucleic acids comprising a sequence encoding an ERGisoform (e.g., ERG8, EPC1, EPC2, a transcript from the prostatecancer-specific promoter, ERG1, ERG2, or ERG3) protein or functionalderivative thereof, are administered to promote ERG function, by way ofgene therapy. Alternatively, nucleic acids comprising an siRNA, shRNA,or antisense of ERG8, EPC1, EPC2, a transcript from the prostatecancer-specific promoter, ERG1, ERG2, or ERG3 sequence are administeredto antagonize ERG expression or function. Any of the methods for genetherapy available in the art can be used. For specific protocols, seeMorgan, GENE THERAPY PROTOCOLS, 2^(nd) ed., Humana Press (2001). Forgeneral reviews of the methods of gene therapy, see Goldspiel et al.,CLIN PHARMACY 12:488-505 (1993); Wu et al., BIOTHERAPY 3:87-95 (1991);Tolstoshev, ANN REV PHARMACOL TOXICOL 32:573-596 (1993); Mulligan,SCIENCE 260:926-932 (1993); Morgan et al., ANN REV BIOCHEM 62:191-217(1993); and May, TIBTECH 11(5):155-215 (1993). Methods commonly known inthe art of recombinant DNA technology which can be used are described inAusubel et al., eds., CURRENT PROTOCOLS IN M OLEC BIOL, John Wiley &Sons, NY (2004); and Kriegler GENE TRANSFER AND EXPRESSION, A LABORATORYMANUAL, Stockton Press, NY (1990).

In some embodiments, the therapeutic comprises an ERG isoform, such asERG8, EPC1, EPC2, a transcript from the prostate cancer-specificpromoter, ERG1, ERG2, or ERG3, or an antisense of one or more of theseERG isoforms. The nucleic acid is part of a vector that has a regulatorysequence, such as a promoter, operably linked to the ERG isoform codingregion or antisense molecule, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another embodiment, anucleic acid molecule is used in which an ERG isoform e.g., (e.g., ERG8,EPC1, EPC2, a transcript from the prostate cancer-specific promoter,ERG1, ERG2, or ERG3) coding sequence and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe ERG isoform (Koller et al., PROC NATL ACAD SCI USA 86:8932-8935(1989); Zijlstra et al., NATURE 342:435-438 (1989)).

In some embodiments, the nucleic acid to be introduced for purposes ofgene therapy comprises an inducible promoter operably linked to thedesired nucleic acids, such that expression of the nucleic acid iscontrollable by the appropriate inducer of transcription.

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using, a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286, which is incorporated herein byreference), or by direct injection of naked DNA, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, DuPont), orcoating with lipids or cell-surface receptors or transfecting agents,encapsulation in liposomes, microparticles, or microcapsules, or byadministering it in linkage to a peptide which is known to enter thenucleus, by administering it in linkage to a ligand subject toreceptor-mediated endocytosis (see, e.g., Wu et al., J BIOL CHEM262:4429-4432 (1987)). In another embodiment, a nucleic acid-ligandcomplex can be formed in which the ligand comprises a fusogenic viralpeptide to disrupt endosomes, allowing the nucleic acid to avoidlysosomal degradation. In yet another embodiment, the nucleic acid canbe targeted in vivo for cell-specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Pubs. WO 92/06180; WO92/22635; WO92/20316; WO93/14188; WO 93/20221). Alternatively, thenucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression, by homologous recombination (Koller etal., PROC NATL ACAD SCI USA 86:8932-8935 (1989); Zijlstra et al., NATURE342:435-438 (1989)).

In some embodiments, a viral vector that contains an ERG isoform ERG8,EPC1, EPC2, a transcript from the prostate cancer-specific promoter,ERG1, ERG2, or ERG3) nucleic acid or antisense nucleic acid is used. Forexample, a retroviral vector can be used. (Miller et al., METH ENZYMOL217:581-599 (1993)). These retroviral vectors have been modified todelete retroviral sequences that are not necessary for packaging of theviral genome and integration into host cell DNA. The ERG isoform (e.g.,ERG8, EPC1, EPC2, a transcript from the prostate cancer-specificpromoter, ERG1, ERG2, or ERG3) nucleic acid to be used in gene therapyis cloned into the vector, which facilitates delivery of the gene into apatient. More detail about retroviral vectors can be found in Boesen etal., BIOTHERAPY 6:291-302 (1994), which describes the use of aretroviral vector to deliver the MDRL gene to hematopoietic stem cellsin order to make the stem cells more resistant to chemotherapy. Otherreferences illustrating the use of retroviral vectors in gene therapyare: Clowes et al., J CLIN INVEST 93:644-651 (1994); Kiem et al., BLOOD83:1467-1473 (1994); Salmons et al., HUM GENE THER 4:129-141 (1993); andGrossman et al., CURR OPIN GEN DEVEL 3:110-114 (1993).

Other viral vectors that can be used in gene therapy includeadenoviruses, which are capable of infecting non-dividing cells.Kozarsky et al., CURR OPIN GEN DEVEL 3:499-503 (1993) present a reviewof adenovirus-based gene therapy. Bout et al., HUM GENE THER 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,SCIENCE 252:431-434 (1991); Rosenfeld et al., CELL 68:143-155 (1992);and Mastrangeli et al., J CLIN INVEST 91:225-234 (1993).Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., PROC SOC EXP BIOL M ED 204:289-300 (1993)).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient. In this embodiment, the nucleicacid is introduced into a cell prior to administration in vivo of theresulting recombinant cell. Such introduction can be carried out by anymethod known in the art, including but not limited to transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, etc. Numerous techniques are known in the art forthe introduction of foreign genes into cells (see, e.g., Loeffler etal., METH ENZYMOL 217:599-618 (1993); Cohen et al., METH ENZYMOL217:618-644 (1993); Cline, PHARMAC THER 29:69-92 (1985)) and may be usedin accordance with the present invention, provided that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted. The technique should provide for the stable transfer of thenucleic acid to the cell, so that the nucleic acid is expressible by thecell and preferably heritable and expressible by its cell progeny. Theresulting recombinant cells can be delivered to a patient by variousmethods known in the art.

The prostate cancer-specific transcripts encode protein products thatare thought to either directly or indirectly contribute to thedevelopment of the cancerous cell. Accordingly, methods that destabilizethese transcripts can be used to reduce or prevent expression of theencoded protein product, thereby preserving the cell in a non-cancerousstate, or reverting the cell to a non-cancerous phenotype. In someembodiments, therefore, nucleic acids corresponding to ERG8, EPC1, EPC2,ERG1, ERG2, ERG3, or isoforms encoded by transcripts initiated from aprostate cancer-specific promoter (e.g., SEQ ID NO: 7), or a fragmentthereof (such as a fragment comprising at least nucleotides 521 to 650of SEQ ID NO: 7), are used to interfere with the production ortranslation of their corresponding transcript. In some cases, thenucleic acid is the complement of the transcript sequence. In thesecases, the nucleic acids are therapeutic because they modulate thefunction of nucleic acids encoding an ERG isoform, such as ERG8, EPC1,EPC2, ERG1, ERG2, ERG3, or isoforms encoded by transcripts initiatedfrom a prostate cancer-specific promoter, and thereby alter expressionof the encoded isoform.

One method of modulating the function of one or more ERG isoforms is viaRNA interference, for example, using siRNA or shRNA against the ERGisoform. The siRNA is a short double stranded RNA molecule of about18-25 nucleotides that comprises a nucleotide sequence complementary toa region of the target. It can be introduced into a target cell ortissue, for example using an expression plasmid, where it interfereswith the translation of an ERG isoform, such as ERG8, EPC1, EPC2, ERG1,ERG2, ERG3, or isoforms encoded by transcripts initiated from a prostatecancer-specific promoter, such as SEQ ID NO: 7 (or a fragment thereof).RNA interference techniques can be carried out using known methods asdescribed, for example, in published U.S. Patent Applications20060058255, 20040192626, 20040181821, and 20030148519, each of which isincorporated by reference.

Antisense compounds are another class of nucleic acid that is providedby the disclosure for use in modulating the function of nucleic acidmolecules encoding one or more ERG isoforms, thereby modulating theamount of the ERG isoform(s) that is produced. This is accomplished byproviding antisense compounds that hybridize with one or more nucleicacids encoding an ERG isoform to a cell, for example, by using a genetherapy technique. The nucleic acid can be DNA encoding an ERG isoform(e.g., ERG8, EPC1EPC2, ERG1, ERG2, ERG3, or isoforms encoded bytranscripts initiated from a prostate cancer-specific promoter, such asSEQ ID NO: 7), RNA (including pre-mRNA and mRNA) transcribed from suchDNA, and can also be cDNA derived from such RNA. The hybridization of anantisense compound with its target nucleic acid interferes with thenormal function of the nucleic acid. The interference can act at thelevel of replication or transcription of the DNA, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, or catalyticactivity that may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function is themodulation of the expression of an ERG isoform, such as ERG8, EPC1,EPC2, ERG1, ERG2, ERG3, or isoforms encoded by transcripts initiatedfrom a prostate cancer-specific promoter, such as SEQ ID NO: 7 (or afragment thereof, such as a fragment comprising at least nucleotides 521to 650 of SEQ ID NO: 7).

Antisense oligonucleotides are one form of antisense compound. Theseoften comprise from about 8 to about 30 nucleobases (i.e. from about 8to about 30 linked nucleosides). In some embodiments, the antisenseoligonucleotide comprises from about 12 to about 25, from about 15 toabout 22, or from about 18 to about 20 nucleobases. Antisenseoligonucleotides can also comprise modified backbones or non-naturalinternucleoside linkages. Modified oligonucleotides that do not have aphosphorus atom in their internucleoside backbone are also consideredoligonucleotides. Examples of modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonzites, phosphinates, phosphoramidates including3′-amino phosplioramidate and aminoalkylphosphoramidates,thionophosphoiamidates, thionoalkylphosphonates,thionoalkylphosphotriesters, boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′, and backbones formed by morpholino linkages.

Peptide nucleic acid (PNA) compounds are also antisense compounds. In aPNA compound, however, the sugar-backbone of an oligonucleotide isreplaced with an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone.

Antisense compounds, methods for their production, and their use tointerfere with nucleic acid function are well known in the art. Forexample, U.S. Pat. No. 6,054,316, which is incorporated by reference,describes the production of antisense compounds for nucleic acidsencoding Ets-2 and methods of using these antisense compounds. Thesesame methods can be applied to the production of antisense compounds fornucleic acids encoding an ERG isoform, such as ERG8, EPC1, EPC2, ERG1,ERG2, ERG3, or isoforms encoded by transcripts initiated from a prostatecancer-specific promoter, such as SEQ ID NO: 7 (or a fragment thereof,such as a fragment comprising at least nucleotides 521 to 650 of SEQ IDNO: 7).

In addition to therapeutic applications related to inhibition ofexpression of ERG isoforms (e.g., ERG8, EPC1, EPC2, ERG1, ERG2, ERG3, orisoforms encoded by transcripts initiated from a prostatecancer-specific promoter, such as SEQ ID NO: 7), antisense compounds arealso useful in diagnostic and prognostic methods because these compoundshybridize to nucleic acids encoding ERG isoforms, which can be detectedusing art-recognized techniques, such as conjugation of an enzyme to theantisense compound, radiolabelling of the antisense compound, or anyother suitable detection methods. Kits comprising the antisense compoundand a means for detecting it in a sample can also be prepared asdescribed for kits comprising oligonucleotide probes generally.

Antisense modulation of ERG isoform expression can be assayed in avariety of ways known in the art. For example, mRNA levels can bequantitated by, e.g., northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). RNA analysis can beperformed on total cellular RNA or poly(A)+ mRNA. Alternatively or inaddition, levels of the encoded protein can be quantitated in a varietyof ways well known in the art, such as immunoprecipitation, western blotanalysis (immunoblotting), ELISA, or fluorescence-activated cell sorting(FACS).

It is also possible to kill or slow the growth of prostate cancer cellsby delivering to those cells a cytotoxic or cytostatic gene productexpressed under the control of a prostate cancer-specific promoter, suchas the promoter sequence set forth in SEQ ID NO: 7. Truncations andvariation of the nucleotide sequence set forth in SEQ ID NO: 7 can alsobe used, so long as they are sufficient to support expression of anoperatively linked reporter gene in prostate cancer cells. Examplesinclude promoter sequences comprising at least nucleotides 521 to 650,404 to 650, or 138 to 650 of SEQ ID NO: 7. Gene therapy can be used tointroduce a vector comprising the prostate cancer-specific promoteroperably linked to a nucleic acid encoding the cytotoxic or cytostaticprotein into a prostate cancer cell. Such gene therapy methods aredescribed herein. When the prostate cancer-specific promoter is used inthe gene therapy vector, however, the promoter is only active in theprostate cancer cells so that the cytotoxic or cytostatic protein isonly expressed in the prostate cancer cells, irrespective of thecellular range of the gene therapy vector.

There are many different cytotoxic cytostatic proteins that can beexpressed by placing a heterologous gene under the control of a prostatecancer-specific promoter. Examples of such genes include genes encodingbacterial toxins, such as diphtheria toxin, pseudomonas toxin, ricin,cholera toxin, and PE40; tumor suppressor genes, such as APC, CYLD,HIN-1, KRAS2b, p16, p19, p21, p27, p27mt, p53, p57, p73, PTEN, Rb,Uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18,MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-1, zac1,scFV, MMAC1, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, and Gene 21 (NPRL2);genes encoding apoptosis-inducing proteins, such as CD95, caspase-3,Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MST1,bbc3, Sax, BIK, BID, and mda7; and genes encoding drug metabolizingenzymes that convert a pro-drug into a cytotoxic product, such asthymidine kinase from herpes simplex or varicella zoster viruses),cytosine deaminase, nitroreductase, cytochrome p-450 2B1, thymidinephosphorylase, purine nucleoside phosphorylase, alkaline phosphatase,carboxypeptidases A and G2, linamarase, β.-lactamase and xanthineoxidase.

Accordingly, the disclosure provides a method of treating prostatecancer comprising administering to a subject in need thereof anexpression vector comprising a polynucleotide encoding a cytotoxic orcytostatic gene product operably linked to a promoter sequencecomprising SEQ ID NO: 7 or a fragment of the nucleotide sequence setforth in SEQ ID NO: 7 that is sufficient to support expression of anoperatively linked reporter gene in prostate cancer cells, including,for example, a sequence comprising at least nucleotides 521 to 650 ofSEQ ID NO: 7. In another embodiment, the disclosure provides a method ofreducing the growth of a prostate cancer cell comprising administeringto the cell an expression vector comprising a polynucleotide encoding acytotoxic or cytostatic gene product operably linked to a promotersequence comprising SEQ ID NO: 7 or a fragment of the nucleotidesequence set forth in SEQ ID NO: 7 that is sufficient to supportexpression of an operatively linked reporter gene in prostate cancercells, including, for example, a sequence comprising at leastnucleotides 521 to 650 of SEQ ID NO: 7. In either embodiment, thecytotoxic or cytostatic gene product is chosen from bacterial toxins,tumor suppressor gene products, apoptosis-inducing proteins, and drugmetabolizing enzymes that convert a pro-drug into a cytotoxic product.

Another way to kill a prostate cancer cell or to inhibit or slow itsgrowth is by modulating the activity of proteins within the cell. Forexample, an antibody that binds a protein encoded by an ERG isoform canbe used to inhibit or stimulate the function of that protein. In someembodiments, the antibody binds an epitope that is present in proteinsencoded by more than one ERG isoforms. Other embodiments involve anantibody that binds the protein encoded by a particular ERG isoform,such as ERG8, EPC1, EPC2, ERG1, ERG2, ERG3, or an isoform encoded by atranscript initiated from a prostate cancer-specific promoter, such asSEQ ID NO: 7 (or a fragment thereof, such as a fragment comprising atleast nucleotides 521 to 650 of SEQ ID NO: 7). Thus, in one embodimentthe disclosure provides an antibody that binds an epitope comprisingamino acid residues 217 to 220 of SEQ ID NO: 4. In another embodiment,the antibody binds an epitope within or comprising amino acids 28 to 97of SEQ ID NO: 6. The antibody or combination of antibodies can beexpressed intracellularly using gene therapy, as described herein. Inanother example, the antibody binds an epitope within or comprisingamino acid residues 28 to 97 of SEQ ID NO: 6, and it also hinds theprotein consisting of SEQ ID NO: 6.

These various antibodies can be produced using techniques known in theart. For example, the protein(s) encoded by one or more ERG isoformERG8, EPC1, EPC2, ERG1, ERG2, ERG3, or an isoform encoded by atranscript initiated from a prostate cancer-specific promoter, such asSEQ ID NO: 7) can be used as an immunogen and then one or moreantibodies with the desired specificity and functional properties can beselected. Such antibodies include, but are not limited to, polyclonalantibodies, monoclonal antibodies, chimeric antibodies, single chainantibodies, and antibody fragments. The antibodies may be from mice,rats rabbits, hamsters, goats, llamas, humans, or other species.

Various procedures known in the art can be used for the production ofpolyclonal antibodies to one or more epitopes of a secreted protein.Rabbits, mice, rats, goats, llamas, etc. can be immunized with thenative protein, a synthetic version of the protein, or a derivative(e.g., fragment) of the protein. Various adjuvants may be used toincrease the immunological response, depending on the host species.Examples of adjuvants include, but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum.

For the preparation of monoclonal antibodies, any of a number ofart-recognized techniques can be utilized. For example, monoclonalantibodies can be produced using the hybridoma technique (e.g., Kohleret al., NATURE 256:495-97 (1975); and as described in Harlow et al.,eds., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory,(1988); Colligan et al., eds., CURRENT PROTOCOLS IN I MMUNOLOGY, Chpt.2, John Wiley & Sons, Inc. (2006)). Antibodies can also be producedusing recombinant DNA methods (e.g., U.S. Pat. No. 4,810,567) or usingphage display antibody libraries (e.g., Clackson et al., NATURE 352:624-28 (1991); Marks et al., J MOL BIOL 222: 581-97 (1991)). If desired,chimeric antibodies can be produced using methods known in the art(e.g., Morrison et al., PROC NATL ACAD SCI USA 81:6851-55 (1994);Neuberger et al., NATURE 312:604-08 (1984); Takeda et al., NATURE314:452-54 (1985)). Single chain antibodies can also be produced (e.g.,U.S. Pat. No. 4,946,778). Human antibodies can be prepared using humanhybridomas (Cote et al., PROC NATL ACAD SCI USA 80:2026-30 (1983)), bytransforming human B cells with EBV virus in vitro (Cole et al.,MONOCLONAL ANTIBODES AND CANCER THERAPY, Alan R. Liss, pp. 77-96(1985)), or by preparing hybridomas from animals transgenic for one ormore human immunoglobulin genes (e.g., U.S. Pat. No. 5,939,598). Amonoclonal antibody can be readily expressed using its encoding DNAsequence(s), and methods for such expression, including gene therapymethods, are well known in the art.

Antibody fragments can also be generated using known techniques.Fragments include but are not limited to F(ab′)₂ fragments, which can beproduced by pepsin digestion of the antibody molecule; Fab′ fragments,which can be generated by reducing the disulfide bridges of the F(ab′)₂fragment; Fab fragments, which can be generated by treating the antibodymolecule with papain and a reducing agent; and Fv fragments, includingsingle chain Fv (scFv) fragments.

Following the production of antibodies by, for example, hybridomatechnology, screening for the desired antibody can be accomplished bytechniques known in the art, e.g., ELISA, and involve no more thanroutine techniques (e.g., Harlow et al., eds., ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, (1988); Colligan et al., eds.,CURRENT PROTOCOLS IN IMMUNOLOGY, Chpt. 2, John Wiley & Sons, Inc.,2006). Thus, an antibody can be selected that binds a linear epitope ora conformational epitope. An antibody also can be selected for theproperty of binding both to a polypeptide fragment of a larger protein,and to the intact (e.g., full length or wild-type) protein.

When it is necessary to produce an antibody to a protein encoded by anERG isoform (e.g., ERG8, EPC1, EPC2, ERG1, ERG2, ERG3, or an isoformencoded by a transcript initiated from a prostate cancer-specificpromoter, such as SEQ ID NO: 7), the protein, its fragment, or otherderivative, can be produced using standard techniques. Methods ofmanipulating nucleic acids to express proteins are well known in theart, and include those described in MOLEC CLONING, A LABORATORY MANUAL(2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor) andCURRENT PROTOCOLS IN MOLEC BIOL (eds. Ausubel, Brent, Kingston, More,Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, N.Y.,NY, (1992)).

Generally, in order to express the protein encoded by an ERG isoform(e.g., ERG8, EPC1, EPC2, ERG1, ERG2, ERG3, or an isoform encoded by atranscript initiated from a prostate cancer-specific promoter, such asSEQ ID NO: 7), a suitable cell line is transformed with a DNA sequenceencoding that protein under the control of known regulatory sequences.The transformed host cells are cultured and the protein recovered andisolated from the culture medium. The isolated expressed proteins aresubstantially free from other proteins with which they are co-producedas well as from other contaminants. Suitable cells or cell lines may bemammalian cells, such as Chinese hamster ovary cells (CHO), the monkeykidney COS-1 cell line, or mammalian CV-1 cells. The selection ofsuitable mammalian host cells and methods for transformation, culturing,amplification, screening, product production and purification are knownin the art. (See, e.g., Gething and Sambrook, NATURE 293:620-625 (1981);Kaufman et al., MOL CELL BIOL 5(7):1750-1759 (1985); Howley et al., U.S.Pat. No. 4,419,446.))

Bacterial cells may also be used as suitable hosts for expression of thesecreted proteins. For example, the various strains of E. coli (e.g.,HB101, MC1061) are well-known as host cells in the field ofbiotechnology. Various strains of a B. subtilis, Pseudomonas, otherbacilli and the like may also be used. For expression of a protein inbacterial cells, DNA encoding the propeptide is generally not necessary.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the secreted proteinbiomarkers. Additionally, where desired, insect cells may be utilized ashost cells in the method of the present invention. See, e.g., Miller etal., GENETIC ENGINEERING 8:277-298, Plenum Press (1986).

In some embodiments, the protein encoded by an ERG isoforms (e.g., ERG8,EPC1, EPC2, ERG1, ERG2, ERG3, or an isoform encoded by a transcriptinitiated from a prostate cancer-specific promoter, such as SEQ ID NO:7) is expressed using a vector that contains a full length DNA sequenceencoding the protein and appropriate expression control sequences.Expression control sequences for such vectors are known to those skilledin the art and may be selected depending upon the host cells. Suchselection is routine. In other embodiments, the secreted proteinbiomarker is expressed as a fusion protein comprising the proteinsequence of the biomarker and, for example, a tag to stabilize theresulting fusion protein or to simplify purification of the secretedprotein biomarker. Such tags are known in the art. Representativeexamples include sequences which encode a series of histidine residues,the epitope tag FLAG, the Herpes simplex glycoprotein D,beta-galactosidase, maltose binding protein, streptavidin tag orglutathione S-transferase.

In some embodiments, therefore, it is desirable that protein expressionof ERG8, EPC1, EPC2, ERG1, ERG2, ERG3, or an isoform encoded by atranscript initiated from a prostate cancer-specific promoter, such asSEQ ID NO: 7, is entirely by an in vitro method. Of course, as alreadydiscussed in other embodiments it is desirable that the proteinexpression occurs in viva

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. Moreover, advantages described in the body of thespecification, if not included in the claims, are not per se limitationsto the claimed invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Moreover, it mustbe understood that the invention is not limited to the particularembodiments described, as such may, of course, vary. Further, theterminology used to describe particular embodiments is not intended tobe limiting, since the scope of the present invention will be limitedonly by its claims. The claims do not encompass embodiments in thepublic domain.

With respect to ranges of values, the invention encompasses eachintervening value between the upper and lower limits of the range to atleast a tenth of the lower limit's unit, unless the context clearlyindicates otherwise. Further, the invention encompasses any other statedintervening values. Moreover, the invention also encompasses rangesexcluding either or both of the upper and lower limits of the range,unless specifically excluded from the stated range.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest the invention. Further, all publications mentioned herein areincorporated by reference in their entireties.

It must be noted that, as used herein and in the appended claims, thesingular forms “a,” “or,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asubject polypeptide” includes a plurality of such polypeptides andreference to “the agent” includes reference to one or more agents andequivalents thereof known to those skilled in the art, and so forth.

Further, all numbers expressing quantities of ingredients, reactionconditions, % purity, polypeptide and polynucleotide lengths, and soforth, used in the specification and claims, are modified by the term“about,” unless otherwise indicated. Accordingly, the numericalparameters set forth in the specification and claims are approximationsthat may vary depending upon the desired properties of the presentinvention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits, applying ordinary roundingtechniques. Nonetheless, the numerical values set forth in the specificexamples are reported as precisely as possible. Any numerical value,however, inherently contains certain errors from the standard deviationof its experimental measurement.

The specification is most thoroughly understood in light of thereferences cited herein. Each of these references is hereby incorporatedby the reference in its entirety.

ERG Isoform Identification and Expression in Prostate Cancer Tissue andCell Lines EXAMPLE 1 Identification of ERG8

ERG1 is the most commonly overexpressed proto-oncogene in malignantprostatic tissue. (Petrovics et al., ONCOGENE 24: 3847-52 (2005).) Thisoverexpression may be due to the fusion of the TMPRSS2 gene with the ERGgene. (Tomlins et al., SCIENCE 310:844-48 (2005).) Alternative splicinggenerates multiple ERG isoforms. (Owczarek et al., GENE 324:65-77(2004).) Thus, it is possible that other isoforms of ERG are alsooverexpressed, or are selectively expressed, in prostate cancer.

In an initial experiment, we sought to detect the ERG8 isoform ire cDNAderived from laser microdissected (LCM) prostate tumor cells. The cDNAwas amplified using a primer pair from the genomic sequence of exon 1 ofthe TMPRSS2 gene (primer p2178: 5′-TAGGCGCGAGCTAAGCAGGAG-3′ SEQ ID NO:8) and from the ERG coding sequence (primer p2220:5′-CCAGGATGCCTTCTTTGCCCATC-3′-SEQ ID NO: 9). The TMPRSS2 gene is oftenfused to the ERG gene in prostate cancer. The p2718 primer correspondsto nucleotides 1 to 21 of SEQ ID NO: 1, while p2220 corresponds to thereverse complement of nucleotides 1042 to 1062 of SEQ ID NO: 1. Thisprimer pair resulted in a PCR product and sequencing confirmed it wasERG8.

ERG8 cDNA can also be amplified using primer pairs directed to ERG8specific regions of SEQ ID NO: 30 and SEQ ID NO: 46. For example, theprimer pair

Mid-E8N-F: (SEQ ID NO: 35) 5′-GGCATTTCCCAGAAGGGTAT-3′ Mid-E8N-R:(SEQ ID NO: 36) 5′-CATCAGCTGTGAGGCTGGTA-3′amplifies nucleotides 1744-1848 of SEQ ID NO: 30 and nucleotides942-1056 of SEQ ID NO: 46. The primer pair

Down-E8N-F: (SEQ ID NO: 37) 5′-AGTGACTTCAGGCTGGCAAT-3′ Down-E8N-R:(SEQ ID NO: 38) 5′-GCTAAGCCTTTCCATCATGC-3′amplifies nucleotides 2087-2176 of SEQ ID NO: 30 and nucleotides1285-1374 of SEQ ID NO: 46. The primer pair

PoA-E8N-F: (SEQ ID NO: 39) 5′-ACCCAGTGCCTAGAGTCTGC-3′ PoA-E8N-R:(SEQ ID NO: 40) 5′-AAGCTTGAGTCAAACATGAATTTCT-3′amplifies nucleotides 2253-2343 of SEQ ID NO: 30 and nucleotides1451-1541 of SEQ ID NO: 46.

The novel full-length ERG8 cDNA of the invention was derived from totalRNA isolated from the prostate tissue of a prostate cancer patient. ERGand TMPRSS2 positive cDNA clones were amplified from the CPDR humanprostate cancer Lambda ZAP phage cDNA library. To amplify the clones, 12μl of 2× Pful enzyme/buffer mixture (Stratagene, La Jolla, Calif.) wascombined with 7 μl of HPLC-grade water, 2 μl (5 pmol) T3 primer, 2 μl (5pmol) T7 primer (both primers from Lofstrand Labs, Bendigo, Australia),and 1 μl ERG-positive Lambda phage clones identified from the library.The reaction conditions were the following: 35 cycles at 94° C., for 1min, 50° C. for 1 min, and 72° C. for 4 min. The thermal cycles werefollowed by a 7 in incubation at 72° C. and storage at 4° C. The PCRproduct of this reaction was purified using Performa Spin Columns-33617(Edge Biosystems, Gaithersburg, Md.).

Nucleotide sequencing was performed by primer extension using the BioDyeSequencing method (Applied Biosystems, Foster City, Calif.). Four μl ofBig Dye Terminator, 2 μl of 5× Sequencing Buffer, 2 μl (5 pmol) of T7sequencing primer, 2 μl of the PCR product (the template), and 10 μl ofwater were added into a total reaction volume of 20 μl. The mixture wasincubated at 96° C., for 60 sec, followed by 25 cycles of 96° C. for 10sec, 50° C. for 5 sec and 60° C. for 4 min. The DNA sequencing reactionmixture was then analyzed on an ABI 3700 Sequence Analyzer. The observedDNA sequences were compared to NCBI databases searching for matches to(DNA sequences and expressed sequence tags (EST). The analysis revealeda 100% match in the overlapping region of the identified clone with theNCBI AY204742 ERG8 cDNA.

The sequence was confirmed with the following internal primers:

(SEQ ID NO: 31) 5′-GAGCGCCGCCTGGAGCGCGGCAG-3′ (SEQ ID NO: 32)5′-TTCAGAAAGACAGATGGGC-3′ (SEQ ID NO: 33) 5′-CACGGATGGTATGATGGTG-3′The results of the sequencing demonstrated that the DNA sequences of theidentified prostate cancer cDNA clones extend beyond the 3′ end of theAY204742 ERG8 sequence. The primer

(SEQ ID NO: 34) 5′-AGCAATACAGGGCGTAGGAG-3′was designed to cover the 3′ end of the cDNA sequences reaching theLambda ZAP polycloning sequences.

The entire ERG8 nucleotide sequence is shown as SEQ ID NO: 30 in FIG. 1.It corresponds to nucleotides 150-1460 of NCBI Accession No. AY204742,with the exception that the adenosine residues identified at nucleotidepositions 1455 and 1459 in AY204742, are identified herein as thymidineat position 1455 and cytosine at position 1459. The ERG8 sequence of SEQID NO: 30 comprises a 20-mer polyA tail at nucleotides 2416-2435 (FIG.1).

EXAMPLE 2 ERG8 is Selectively Expressed in Prostate Cancer Tissue

We then undertook a more thorough examination of the expression ratiosof the ERG1, ERG2, ERG3, and ERG8 isoforms in normal prostate cells andin the prostate cancer-derived cell line VCaP (American Type CultureCollection (“ATCC”), Rockville, Md.). We isolated mRNA from normalprostate of 11 individuals and from prostate cancer-derived VCaP cells,respectively. After converting the mRNA to cDNA, we assessed ERG isoformratios by comparing the intensities of isoform-specific PCR products ina semi-quantitative multiplex PCR approach. FIG. 2 presents the resultsof the multiplex PCR analysis. The ERG primers used for the PCR were asfollows: p2192 (exon 9): 5′-ACCGTTGGGATGAACTACGGCA-3′ (SEQ ID NO: 10,which corresponds to nucleotides 352 to 373 of SEQ ID NO: 1); p2220:(ERG8 specific): 5′-CCAGGATGCCTTCTTTGCCCATC-3′ (SEQ ID NO: 11, whichcorresponds to the reverse complement of nucleotides 1042 to 1064 of SEQID NO: 1); p2207: (exon 16): 5′-CCCTCCCAAGAGTCTTTGGATCTC-3′ (SEQ ID NO:12); p2197: (axon 15): 5′-CCTGGATTTGCAAGGCGGCTACT-3′ (SEQ IQ NO: 13);and p2198: (axon 11): 5′-CTCTCCACGGTTAATGCATGCTAG-3′ (SEQ ID NO: 14,which corresponds to nucleotides 699 to 722 of SEQ ID NO: 1).

Primer pair p2192-p2220 results in a 713 bp PCR product when ERG8 ispresent The combination of primers p2192 and p2207 amplifies ˜1300 bpproducts representing ERG isoforms 1, 2 and 3. When p2192 (Exon 9) ispaired with primer p2197 (Exon 15), that primer combination amplifiesone or more of ERG isoforms 1, 2 and 3. Primer pair p2198-p2220 is alsospecific to the ERG8 isoform, and this primer pair amplifies a 366 bpPCR product when ERG8 is present. The combination of p2198 (Exon 11) andp2207 (Exon 16) results in a 959 bp product detecting ERG isoforms 1, 2and 3, while the p2198-p2197 combination yields products of 279 bp(isoform 3) and 207 bp (isoforms 1 and 2).

In FIG. 2, the normal prostate samples are labeled “NP”, while samplesusing the VCaP cells are labeled “VC”. ERG8 is the predominant isoformdetected in VCaP cells (FIG. 2, right photograph, upper arrow). It isalso present at higher levels than ERG1 and ERG2 in normal prostate, butits level in normal prostate is comparable to that of ERG3.

We have also assayed the expression of ERG8 transcripts in RNA specimensextracted from laser microdissected (LCM) tumor and benign epithelialcells of 14 individual prostate cancer patients. Primers specificallyrecognizing the ERG8 isoform (p2198-p2220) were used together with GAPDHprimers as internal controls in the same RT-PCR reaction tubes. FIG. 3shows a photograph of a PCR gel with data for eight of the patients.Tumor cell samples are labeled “T”, while the benign epithelial cellsamples from each patient are labeled “N”. ERG8 expression was detectedin the tumor cell samples of 11 of the 14 prostate cancer patientstested. We did not detect ERG8 expression in the benign cells of anypatient in this cohort. Thus, ERG8 isoform detection indicates thepresence of cells with a cancerous phenotype. In FIG. 3, the level ofERG8 is below the detection limit in the normal samples, which includeonly epithelial cells. The difference between ERG8 detection in FIG. 2and FIG. 3, therefore, can be explained by the presence of the othercell types (e.g., stroma and endothelial cells) included in the pooledprostate tissue used for the analysis in FIG. 2.

Interestingly, the ERG8 transcript (SEQ ID NO: 1, SEQ ID NO: 30, and SEQID NO: 46) is a fusion between TMPRSS2 and ERG8. The open reading frame,however, is entirely encoded by the ERG8 sequence (nucleotides 75 to1168 of SEQ ID NO: 1 and SEQ ID NO: 30). The encoded protein (SEQ ID NO:2), therefore, does not contain any amino acid sequences from TMPRSS2.

Cancer cells gain growth advantage by activating cell growth promotinggenes and by silencing inhibitory genes of cell proliferation. Certaingenes in these cell growth or proliferation pathways may producealternative transcript that counteract the function of naturaltranscriptional products. In the case of ERG8, the encoded-proteinproduct lacks the DNA binding domain of for example, ERG1 and ERG2, butit retains the entire protein-protein interaction domain. Overexpressionof ERG8 in the context of prostate cancer, therefore, likely results inthe functional nullification of protein interaction partners of ERG1 andERG2, resulting in a dominant negative effect. ERG8 could also representan oncogenic “gain of function” isoform.

The finding that ERG8 is selectively expressed in prostate cancer cellsprovides a powerful therapeutic option, as this oncogenic ERG8 productcan be inhibited by selective targeting through its distinct 3′sequences. This selective targeting for cancer therapy can beaccomplished using siRNA, shRNA, and other nucleic acid-based productscapable of targeting the ERG8-specific sequence. At the protein level,antibodies and therapeutic agents such as small inhibitory peptides canbe used to inhibit the activity of the protein produced by ERG8 or totarget that protein for degradation. Moreover, ERG8 can differentiatetumor cells from normal epithelial cells in prostate specimens.Accordingly, detection of ERG8 using, for example, amplification primersor hybridization-based methods, can also be used to diagnose andprognose prostate cancer.

EXAMPLE 3 EPC1 and EPC are Newly Identified Transcripts that areSelectively Expressed in Prostate Cancer Tissue

To identify tumor specific ERG transcripts, we pooled prostate tumortissue samples from six patients and extracted total RNA. PolyadenylatedRNA (mRNA) was then isolated, converted into cDNA, and packaged into theLambda Zap express system (Stratagene) to obtain a bacteriophagelibrary. We screened phage plaques by hybridization of radioactivelylabeled ERG2 probes. The ERG2 sequence includes exons used by all otherERG isoforms; accordingly, it can be used as a general ERG probe.Hybridization was performed with 1×10⁶ cpm ³²P-radiolabelled human ERG2cDNA/ml hybridization solution at 65° C. for overnight. Followinghybridization, the membranes were washed sequentially with 2×SSCsupplemented with 01% SDS, then 0.2×SSC supplemented with 0.1% SDS, at65° C. Before we isolated DNA for sequencing, we subjected hybridizationpositive clones to two more rounds of plaque screening to obtain singleplaques.

Two clones yielded novel ERG isoform transcripts. Each clone has aunique 3′ sequence. Because these ERG transcripts have only beenobserved in prostate cancer tissue, we called the clones “EPC1” and“EPC2” for ERG Prostate Cancer-Specific Isoform 1 and 2.

The nucleic acid sequence of the EPC1 clone is set forth in SEQ ID NO:3. This transcript is also a fusion between exons of TMPRSS2 and ERG.The TMPRSS2 derived sequence occurs at the 5′ end upstream of theinitiation methionine (ATG at position 140 to 142 of SEQ ID NO: 3). Thelast four carboxy-terminal amino acids of the EPC1 amino acid sequence(SEQ ID NO: 4) are not found in any ERG exon, and they appear to bederived from an ERG intronic sequence. The unique 3′ end of EPC1corresponds to nucleotides 788 to 1019 of SEQ ID NO: 3 and can be usedin both nucleic acid (e.g., amplification and hybridization-based) andprotein (e.g., antibody-based) detection methods for the detection ofcancer cells or precancerous cells in specimens and biofluids.

The nucleic acid sequence of the EPC2 done is set forth in SEQ ID NO: 5.The amino acid sequence of EPC2 is set forth in SEQ ID NO: 6. The unique3′ sequence corresponds to nucleotides 127 to 807 of SEQ ID NO: 5. The5′ end corresponds to sequences within ERG exon 10, and the sequenceappears to continue into the adjacent 3′ exon without splicing,resulting in a unique transcript sequence.

We next investigated the expression of EPC1 transcripts in RNA specimensextracted from laser microdissected (LCM) tumor and benign epithelialcells of 14 prostate cancer patients using RT-PCR. We selected primersspecifically recognizing the EPC1 isoform (p2301-p2302) and used themtogether with GAPDH primers (p2135-p2144) as internal controls in thesame reaction tubes. The EPC1 primer sequences were: p2302:5′-CAGAAAGCAGCCTTCCCTTA-3′ (SEQ ID NO: 15, corresponding to nucleotides820 to 839 of SEQ ID NO: 3); and p2301: 5′-TTGATAATAGAGCATCAGACTTCCA-3(SEQ ID NO: 16, corresponding to the reverse complement of nucleotides953 to 977 of SEQ ID NO: 3).

FIG. 4 shows a photograph of a PCR gel with data for five of the 14patients. Tumor cell samples are labeled “T,” while the benignepithelial cell samples from each patient are labeled “N.” EPC1expression was measured, along with the control gene GAPDH. EPC1expression detected in the tumor cells of 11 of the 14 prostate cancerpatients tested. In seven patients, EPC1 expression could be detectedonly in their prostate tumor cells, while in four patients, EPC1expression could be detected in both their tumor and benign epithelialcells. In those instance where EPC1 was detected in both tumor andbenign epithelial cells, EPC1 expression was increased in tumor cellsrelative to benign epithelial cells.

EPC1 and EPC2 are ERG isoforms that are uniquely expressed in cancerousprostate. At the transcript level, the 3′ end of each transcript isunique and distinct from all known ERG isoforms. It may betherapeutically beneficial to degrade EPC1 and/or EPC2 mRNA (e.g., usingsiRNA or shRNA) or to inhibit the EPC1 and/or EPC2 protein by usingantibodies raised against each distinct C-terminal region.

EXAMPLE 4 The ERG8 and EPC1 Isoforms are Abundantly Expressed

In order to compare the relative abundance of ERG8 and EPC1 isoforms tothat of ERG1, we prepared samples from prostate cancer-derived VCaPcells as well as from microdissected tumor cells from prostate cancerpatients. We then used quantitative PCR to determine the copy numbersusing primer pairs specific for EPC1 ERG8, and for a sequence in commonbetween ERG1 and ERG2. The positions of the various primers and thedomain structure of the ERG isoforms are shown in Figure A. In theschematic diagram, “TM” denotes TMPRSS2 and the boxes numbered 8-16correspond to exons, numbered according to Owczarek et al., GENE324:65-77 (2004)). The ERG8 specific primers and probe were as follows:

ERG8 forward primer: (SEQ ID NO: 17) TTCAGAAAGACAGATGGGCAAA;ERG8 reverse primer: (SEQ ID NO: 18) GTTCAAAAGTCGGCCTATTCCTAA;ERG8 probe: (SEQ ID NO: 19) AAGGCATCCTGGATGCCTGGCA; EPC1 forward primer:(SEQ ID NO: 20) GCACTTCTGCCAAGCATATGAGT; EPC1 reverse primer:(SEQ ID NO: 21) CGCTGATCATTTCAACACCCT; EPC1 probe: (SEQ ID NO: 22)TGCCTTGAAGATCAAAGTCAAAGAGAAATGGA; ERG1/2 Ex 11-13 forward primer:(SEQ ID NO: 23) TTCAGATGATGTTGATAAAGCCTTACA;ERG1/2 Ex 11-13 reverse primer: (SEQ ID NO: 24) TCCAGGCTGATCTCCTGGG;ERG1/2 Ex 11-13 probe: (SEQ ID NO: 25) ATGCATGCTAGAAACACAGATTTACCAT.

The number of copies of different ERG isoforms was determined in VCaPcells by Tag Man QRT-PCR using the specific primers and probes shown inFIG. 5A and the results are shown in FIG. 5B. Plasmid constructscomprising a target gene (different ERG isoforms) insert were used togenerate standard dilution series in which the copy number of plasmidsin the dilution series is known. A formula was derived from the standardcurve to correlate the Ct value with the target gene copy number. Usingthis formula and the standard curve, we calculated the copy numbers ofthe target genes in the samples. As shown in FIG. 5B, the copy number ofboth ERG8 and EPC1 is two-fold or more greater than the copy number ofthe combination of ERG1 and ERG2 in VCaP cells. In addition,microdissected tumor cells of nine of ten prostate cancer patientsexhibited a higher copy number for ERG8 than for the ERG1 and ERG2combination (FIG. 5C). These data indicate that the ERG8 and EPC1isoforms are abundantly expressed and accordingly provide potentialtargets in diagnostic and prognostic applications.

EXAMPLE 5 Combined Detection of ERG8 and EPC1 is Inclusive of allTMPRSS2-ERG Fusions Examined and Results in a Robust Detection Systemfor Prostate Cancer

Our finding that ERG8 is overexpressed in prostate cancer and that EPC1is selectively expressed in prostate cancer tissue can be used todevelop a particularly robust diagnostic and prognostic assays becausethese two genes possess unique 3′ ends The 3′ end of an mRNA transcriptis relatively resistant to degradation compared to its 5′ end, making itpossible to detect sequences near the 3′ end in clinical samples thatmight be difficult to detect if they were located toward the 5′ end ofthe sequence. Thus, although one mechanism of over or selectiveexpression of ERG8, EPC1, and EPC2 may involve a 5′ fusion to TMPRSS2,the 3′ portion of the ERG8, EPC1, and EPC2 sequences should be morestable and readily detectable in clinical samples than the 5′ TMPRSS2sequence. As a result, detecting the 3′ end of ERG8, EPC1, and EPC2transcripts can reduce false negatives compared to detecting 5′sequences such as the 5′ TMPRSS2 sequence in TMPRSS2-ERG fusiontranscripts. In addition, biofluids, such as urine, serum, plasma,saliva, and prostatic fluid that are easier and cheaper to obtain butmore prone to mRNA degradation can be used to detect 3′ sequences ofERG8, EPC1, and EPC2.

Accordingly, we developed a simple PCR assay that detects aberrantexpression due to any type of ERG fusion event. We tested an assay thatutilizes three pairs of PCR primers. Namely, we used p2302 (SEQ ID NO:15) and p2301 (SEQ ID NO: 16) to detect EPC1; p2220 (SEQ ID NO: 11) andp2198 (SEQ ID NO: 14) to detect ERG8; and p2236 and p2237, described inPetrovics et al., ONCOGENE 24:3847-3852 (2005), to detect the 3′ UTR ofERG1/2. These primer combinations detect sequences in the 3′ end thatare retained to any 5′ fusion, such as with TMPRSS2, and that arerelatively resistant to degradation.

We used these three primer pairs to test the presence or absence of ERGisoforms LCM selected prostate cancer cells. We divided the samples intotwo groups, based upon whether we could detect a TMPRSS2-ERG fusiontranscript. Table 1 presents the results.

TABLE 1 FP ERGfusionA ERGfusionA EPC1 ERG8 ERG1 Combined Signal FP3470.865 Yes T T YES FP411 8.07 Yes T T YES FP413 2.52 Yes T T YES FP4735.105 Yes T T T YES FP480 12.005 Yes T no YES FP519 1.44 Yes T T YESFP521 1.07 Yes T T T YES FP554 3.9 Yes T no YES FP564 2.24 Yes T and N TYES FP703 2.66 Yes T and N T YES FP245 −3.305 Yes T and N T YES FP3491.315 Yes T T YES FP355 2.12 Yes T T T YES FP391 2.16 Yes T T YES FP4023.595 Yes T T T (and N) YES FP430 2.77 Yes T T YES FP441 6.2 Yes T T andN no YES FP489 3.6 Yes T T YES FP504 5.435 Yes T YES FP510 4.47 Yes T noYES FP553 2.94 Yes T no YES FP320 No no no FP326 No no no FP346 No no noFP393 No no no FP513 No no no FP535 No no no FP573 No no no FP590 No nono FP598 No no no FP620 No no no FP257 No T and N no YES FP260 No no noFP318 No no FP394 No no no FP446 No no no FP488 No, has fusionC T and NT YES FP491 No no no FP493 No no FP495 No no no FP508 No no FP523 No nono FP575 No, has fusionC T T YES

In Table 1, the “FP” numbers in the left column are the coded specimennumbers. The first 21 samples presented are those in which we coulddetect the “A type” TMPRSS2-ERG fusion transcript. ERG fusion A is themost frequent fusion (95% of all fusion transcripts) and involves fusionof the first exon TMRPSS2 to ERG exon 8. The numeric values in the firstcolumn labeled “ERGfusionA” indicate the threshold cycle numbers,normalized to GAPDH, in a quantitative RT-PCR analysis. In 22 samples,we were unable to detect ERG fusion A, but in two samples, FP488 andFP575, we detected ERG fusion C. Fusion “C” is a rare fusion betweenTMPRSS2 exon 1 and ERG exon 9. In the EPC1, ERG8, and ERG1 columns, “T”indicates detection in tumor cells, “N” indicates detection in normalepithelial cells, and “no” indicates that no signal was detected. The“combined signal” column summarizes the cumulative detection of ERGproducts (“YES”=expression of any of isoforms EPC1, ERG8, or ERG1)

By using this combined signal approach, we could detect an amplificationproduct in all samples bearing an ERG fusion. In addition, in thosesamples in which EPC1 was detected but ERG8 was not (e.g., FP480), westill obtained a combined signal. Although we examined ERG1 expressionin several samples to validate the assay, the results show that it isnot necessary to include ERG1 in the analysis. Instead, the combinedsignal from EPC1 and ERG8 was all that was needed to detect all fusionevents. Accordingly, the combined signal approach can help to minimizefalse negatives that could arise by looking only at a particular ERGtranscript. In addition, we expect that the combined approach can bereadily used in clinical samples, such as biofluids, that would beinappropriate for use with primers for the more 5′ TMPRSS2-ERG fusionevent.

EXAMPLE 6 A Novel ERG Promoter is Activated in Prostate Cancer

To determine whether there are additional alterations that occur in theERG locus in prostate cancer, we systematically evaluated transcriptioninitiation sites within the ERG locus using the 5′ oligocapping method.This information was used to map cancer-specific ERG alternativetranscription start sites. We isolated total RNA from prostate cancertissues of six patients with verified ERG gene rearrangements, pooledthe RNA, then treated it with dephosphatases to degrade non-capped RNAmolecules, thereby enriching 5′ cap protected mRNA molecules. An RNAoligonucleotide adapter was ligated to substitute the 5′ cappingstructure and cDNA was generated by reverse transcription. We then usedthe oligo cap adaptor and internal primers from ERG exon 10 to amplify5′ ERG sequences. In the first amplification we used ERG primer p2181:5′-GGCGTTGTAGCTGGGGGTGAG-3′ (SEQ ID NO: 26). In the secondamplification, we used ERG primer p2268:5′-CAATGAATTCGTCTGTACTCCATAGCGTAGGA-3′ (SEQ ID NO: 27). The resultingPCR products were cloned into the pUC19 vector, then sequenced. DNAsequences indicating transcription initiation sites from ERG sequencesin tumor tissue were matched to the ERG locus and analyzed by generatinga score that represented the frequency of individual transcription startsites within the locus. The 5′ capping frequency map (CapMap) of ERGgene transcripts is shown in FIG. 6. Of the 152: clones sequenced, 137of the 5′ capping clones had novel, prostate cancer-specifictranscription initiation sites within a 23 bp ERG exon 9 region.

In a separate oligocapping experiment, 5′ cap sites were assessed in RNAfrom normal prostates pooled from 11 individuals (AMBION) with anegative prostate cancer diagnosis. In this experiment, we terminatedour analysis after 30 clones because the products were homogenous. Theresults indicated that transcription initiation in normal prostateuniformly occurs in ERG exon 5, in sharp contrast to the multiple exon 9initiation sites we observed in the tumor specimens. Transcriptioninitiation in ERG exon 5 indicates that ERG isoform 3 is expressed innormal prostate. Also, our results suggest that ERG isoforms 1, 2, 5, 6,7, 8 and 9 are either not expressed in normal prostate, or they arepresent only at low levels.

The transcription initiation sites detected in the prostate cancersamples indicated that the central segment of ERG oxen 9 is acancer-specific promoter site. The promoter region is defined as thearea between −520 bp and +130 bp relative to the most 3′ transcriptioninitiation site detected in the mapping experiment. The promotersequence is set forth below:

TCTCTCGCCA GTCTGGAGTG CAGTGGCATG ATCTCAGCTC ACTGCAACCT  50CCACCTCCCG GATTCAAGCA ATTTTCCTGC CTCAGCCTCC TGAGTAGCTG 100GGACTACAGG CATGCCCAGC TAATTTTTGT ATTTTTAGTA GAGACGGGGT 150TTCACCATGT TGGCCAGGAT GGTCTGGATC TCTTGACCTC ATGATCCGCC 200CACCTGGGCA TCCCAAAGTG TTGGGACTAC AGGCATGAGC CACGGCACCC 250CGCCTGTATT TGGCTTTTCA CACTTGTCCT TTCTCCCCCA GTCTCTTCCG 300CCTTGCCCTT CTTTGGTTCT CTCTGTGTAT TGTGAGAAGT CGATGGAGAC 350ATGCTCTTTG ATTGCTGTTA TAATGGAAGA ATATTTCTTC TCCTCCAGGA 400ACTCTCCTGA TGAATGCAGT GTGGCCAAAG GCGGGAAGAT GGTGGGCAGC 450CCAGACACCG TTGGGATGAA CTACGGCAGC TACATGGAGG AGAAGCACAT 500

550 GTCAGGTGCC CACAGCTTCA CTGCCCTCGG CAGATCGCAA CTTCCCCAAG 600GCTAGGCTGA GCCTCAGGGA GCTCTTCTCC CCCACCTGTG GCATTGATCA 650(SEQ ID NO: 7). In the sequence, the most 3′ transcription start sitethat is frequently used is bolded and boxed.

The putative TATA-less promoter is predicted at −20; −40 bp from thetranscription initiation site. Interestingly, there is also a MED(Multiple Elements of Initiation Downstream) in the +130 region, whichmay explain the multiple start sites we observed. We have verified thatthis promoter is functional by operably linking it to the luciferasereporter gene. FIG. 7 demonstrates that the prostate cancer-specificpromoter is able to direct expression of luciferase protein in prostatecancer-derived VCaP cells (light grey bars), which contain a TMPRSS2-ERGfusion, but not in LNCaP cells (dark grey bars). A promoter fragmentfrom −117 to +130 (nucleotides 404 to 650 of SEQ ID NO: 7) yielded thegreatest expression levels in the luciferase assay, followed by the +1to +130 fragment (nucleotides 521 to 650 of SEQ ID NO: 7), then the −383to +130 fragment (nucleotides 138 to 650 of SEQ ID NO: 7).

Activation of a dormant promoter within the ERG gene locus in a cancerspecific manner produces transcripts coding for N-terminal deletionmutants. The encoded protein products lack the protein-proteininteraction domain of wild type ERG. Therefore, expression products ofthis dormant promoter may act as dominant negative or gain-of-functionmolecules. Nucleic acid or protein-based products that manipulate theactivity of this promoter can therefore be used for prostate cancertherapy. In addition, the prostate-specific expression of this promotermeans that expression vectors in which the promoter is operably linkedto a gene encoding a toxin or other inhibitor of cell growth can be usedto selectively express the encoded protein in prostate cancer cell.

EXAMPLE 7 A Regulatory Loop Exists Between ERG and the Androgen Receptor

Gene rearrangements involving the fusion of the androgenreceptor-regulated TMPRSS2 gene promoter and ERG occur at a highfrequency (˜60%) in prostate cancer and are likely to be a direct causeof prostate cell transformation, but the mechanism by which the genomicalteration leads to prostate cancer is thus far unexplained. It islikely that this genomic alteration is responsible for, or at leastcontributes to, the overexpression of ERG1 in prostate cancer. It isalso known that androgen receptor function is central to the growth anddifferentiation of the normal prostate gland. Further, androgen receptordysfunction favors the growth and survival of prostate cancer cells andappears to play a role in prostate cancer progression. It is unclear,however, how these alterations interact to result in prostate cancer.

To investigate whether ERG protein contributes to prostate cancer byinterfering with androgen receptor signaling, we correlated theexpression of TMPRSS2-ERG fusion transcripts with ERG1, androgenreceptor (AR), PSA, and the androgen-regulated gene PMEPA1. LTF was alsoanalyzed as a negative control. The results of this analysis are shownin FIG. 8, which compares the quantitative RT-PCR data for theTMPRSS2-ERG fusion to quantitative RT-PCR data obtained by amplifyingthe 3′ untranslated regions of ERG (“ERG1”).

We next investigated the effect of ERG expression on transcriptionaltargets of the androgen receptor. We introduced two different ERG siRNAsinto the VCaP prostate cancer cell line. The sequence of the siRNAssiRNA-1 (p2094): TGATGTTGATAAAGCCTTA (SEQ ID NO: 28), which targets exon11, and siRNA-2 (p2095): CGACATCCTTCTCTCACAT (SEQ ID NO: 29), whichtargets exon 10. VCaP cells possess a TMPRSS2-ERG fusion and overexpressERG. The results of these experiments are shown in FIG. 9A. Introductionof either siRNA led to the up regulation of NKX3.1 and PSA/KLK3 (FIG.9A, top panel). The upregulation of PSA/KLK3 could also be detected asincreased PSA levels in the VCaP culture supernatant (FIG. 9A, bottompanel).

ERG knockdown also inhibited prostate tumor cell proliferation in vitroand in vivo. FIG. 9B shows the morphology of VCaP cells transfected with50 nM ERG siRNA, compared to controls (“NT”). Experiments were performedin triplicate and the cell morphology monitored on days 1, 4, 6, and 8post transfection (FIG. 9B, top left panel).

ERG siRNA inhibited VCaP cell proliferation, decreasing the number ofcells present in the cultures compared to cells treated with control RNA(FIG. 9B, top right panel). Cells transfected with ERG siRNA werecounted in triplicate at days 1, 2, 4, 6, 8, and 10 post transfection,and observed to grow at a slower rate than control cells (p=0.001).

Fluorescent activated cell sorting (“FACS”) analysis of the cell cycledemonstrated a inhibitory effect of ERG siRNA on the number of cells inS phase, compared to control RNA. The population of cells in S phase andthe distribution of cells in G1, S and G2+M phases was assessed byanalyzing the cells at the indicated time points from three independentexperiments. Phospho-Rb to total Rb ratios were measured at day four byWestern blot assay. ERG siRNA induces a redistribution of the number ofcells in G1, S and G2+M phases, significantly increasing the number ofcells in S phase.

Inhibiting ERG expression with ERG siRNA drastically reduced the growthof VCaP cells injected into severe combined immunodeficient (“SCID”)mice. Male SCID mice (Harlan Sprague-Dawley, National Cancer Institute,Frederick, Md.) 4-6 weeks old and weighing 18 to 20 g, housed in sterilefilter-capped cages, fed and given water ad libitum, were injectedsubcutaneously with VCaP cells. All animal studies were carried outaccording to NIH-approved protocols, in compliance with the Guide forthe Care and Use of Laboratory Animals.

The injected VCaP cells were treated with ERG siRNA or NT control RNA,trypsinized, washed, and three million cells in a volume of 0.2 ml wereinjected into the flanks of the SCID mice. Tumor formation was assessedbi-weekly for up to seven weeks. Tumor growth analysis was performed bydetermining tumor volume (LW2/2), as described by Polverino et al.,CANCER RES 66:8715-21 whereby L and W represent the length and the widthof the tumor.

FIG. 9B, lower right panel, shows the tumor growth at days 35 (p=0.0072)and 42 (p=0.0072). Standard deviation (SD) and average tumor volumes(AVG) at day 42 are shown in the table. All of the animals treated withERG siRNA had a reduced tumor load compared to the control animals andsome had no tumor load (FIG. 9B, lower right panel).

ERG knockdown inhibited VCaP cell growth in complete medium. VCaP cellsseeded into 10 cm tissue culture dishes in medium comprising 10% cFBSwere cultured for three days, then transfected with ERG siRNA or controlRNA. Cells from triplicate experiments were examined on day 1, 4, 6, and8 by microscopy, as shown in FIG. 19.

These data demonstrate a relationship between the in vivo expression ofTMPRSS2-ERG or ERG and the expression of other androgen-regulated genes,such as PSA/KLK3 and NKX3.1. Because downregulation of ERG expressioncorrelates with increased expression of NKX3.1 and PSA/KLK3, thisindicates that these androgen-regulated genes are downregulated byectopic ERG expression. NKX3.1 is a tumor suppressor gene and also atranscriptional target of the androgen receptor. Suppression of NKX3.1by ERG overexpression in prostate cancer cells may therefore interferewith androgen receptor-mediated cell differentiation and negativeregulation of cell growth. A schematic of this model is shown in FIG.10.

As an example of an application for the use of inhibitory molecules intargeting transcripts of the ERG locus for degradation, we used siRNA-1to inhibit ERG expression in VCaP prostate cancer cells. As noted,siRNA-1 targets exon 11, which is found in ERG1, ERG2, ERG3, ERG8, EPC1,and EPC2 transcripts and in the predicted products of the alternativeinternal promoter. VCaP cells respond to androgen hormone treatment,therefore, the effect of ERG inhibition on cell growth can be tested bystimulating the cells with androgen hormone.

To perform the siRNA inhibition, cells were first plated to 30%confluence in 100 mm cell culture dishes. Growth was synchronized byincubating the cells in hormone depleted serum (cFBS) containing mediafor three days. Then the cells were transfected with siRNA-1 andnon-targeting (NT) control siRNA using the lipofectamine 2000 reagent(Invitrogen, Carlsbad, Calif.). After transfection 0.1 nM of R1881synthetic androgen (New England Nuclear, Boston, Mass.) was added to themedia. The cells were incubated for nine days, with a media change everythree days. Cell cultures were then photographed and 100× magnificationof representative view fields were captured.

A microscopic view of VCaP cells is shown in FIG. 11. Cells treated withthe control NT siRNA are shown in FIG. 11A, while FIG. 11B shows cellstreated with siRNA-1. VCaP cells treated with siRNA-1 exhibited a robustreduction in cell numbers. In addition, striking changes in cellmorphology were also apparent. Thus, we were able to show that siRNA-1treatment inhibited androgen stimulated growth of VCaP cells.

Taken together, these data suggest that there is a regulatory loopbetween ERG and the androgen receptor and that negative regulation ofthe androgen receptor by ERG may contribute to prostate tumorigenesis.Accordingly, there are several therapeutic interventions that can beapplied in an early stage prostate cancer (such as well to moderatelydifferentiated tumors) harboring a TMPRSS2-ERG fusion or ERGoverexpression. For example, ERG-siRNA, shRNA, or other small moleculescan be used to reduce ERG expression in early stage prostate cancer,which is the most common stage of prostate cancer identified in post-PSAscreening era. Alternatively or in addition, the androgen receptor canbe selectively inhibited with beneficial effects.

It should be again noted that in the context of therapeuticinterventions, any mention of “ERG” includes not only ERG8, EPC1, EPC2,and transcript products from the prostate cancer-specific promoterdescribed herein, but also ERG1, ERG2, and ERG3, as well as theircombinations, unless specifically indicated to the contrary by contextor by an explicit exclusion of one or more of those isoforms. Thus,although we have exemplified inhibition of androgen-stimulated growthwith an siRNA specific for exon 11, which is shared by ERG1, ERG2, ERG3,ERG8, EPC1 and EPC2 transcripts, siRNA, shRNA, or other small moleculeinhibitors targeted to only one, or any combination of more than one. ofthose isoforms may also be employed. Such siRNA, shRNA, or otherinhibitors that are specific for only one of ERG1, ERG2, ERG3, ERG8,EPC1, EPC2, or a transcript product from the prostate cancer-specificpromoter, or that inhibit combinations of those isoforms, can bedesigned using the sequence data provided elsewhere in the Examples andmay include the various primer and probe sequences mentioned.

For example, ERG8 gene expression can be blocked by targeting ERG8specific nucleotide sequences with inhibitory nucleic acids. Examples ofsense-strand specific sites suitable for targeting are:

(SEQ ID NO: 41) 5′-GGAACCACTTCTAGCAATA-3′ (SEQ ID NO: 42)5′-CGAATAATGAGCAGGGAGA-3′ (SEQ ID NO: 43) 5′-CCAGGGAGCTAAAGAGAAT-3′(SEQ ID NO: 44) 5′-CTGGGAAGCATGATGGAAA-3′ (SEQ ID NO: 45)5′-GACTCAAGCTTTAGAGATT-3′SEQ ID NO: 41 corresponds to nucleotides 1595-1613 of SEQ ID NO: 30; SEQID NO: 42 corresponds to nucleotides 1722-1740 of SEQ ID NO: 30: SEQ IDNO: 43 corresponds to nucleotides 2013-2031 of SEQ ID NO: 30; SEQ ID NO:44 corresponds to nucleotides 2150-2168 of SEQ ID NO: 30; and SEQ ID NO:46 corresponds to nucleotides 2334-2352 of SEQ ID NO: 30.

Progressive tumors that do not express ERG, or express ERG only at lowlevels, reflect an escape from an intact androgen receptor signalingnetwork. These tumors may be treated by selective upregulation ofandrogen-regulated genes (e.g., tumor suppressors or celldifferentiation and growth inhibitors, such as NKX3.1 and PMEPA1), so asto restore the protective component of the feedback regulation betweenERG and the androgen receptor.

EXAMPLE 8 The Androgen Receptor Function Index

The readout of androgen receptor (“AR”) regulated genes ultimatelyreflects the status of in vivo AR function (ARF) in primary prostatecancer tissue, and consequently carries important information regardingprognosis and rational therapeutic decision making. Assessing the statusof AR function in prostate cancer samples can provide earl warning signsof androgen independence (van Gils et al., EUR UROL 48(6):1031-41(2005)). Well characterized, annotated, and preserved human tissues(with long term follow-up data) from the CPDR Biospecimen Bank were usedin high throughput screens to identify and validate prostate cancerbiomarker genes.

In recent years, we have analyzed cell type specific gene expressionfrom microdissected matched tumor and benign prostate epithelial cells.We found a general decrease in androgen regulated gene expression withprostate cancer progression. (Petrovics et al., ONCOGENE 24:3847-52(2005).) Others have also recently noted a signature of attenuated ARfunction in late stage, especially in metastatic prostate cancer inhuman specimens (Tomlins et al., NAT GENET 39(1):41-51 (2007)), as wellas in a xenograft model system (Hendriksen et al., CANCER RES66(10):5012-20 (2006)). As part of a 12-gene panel, PSA was found to beunderexpressed in aggressive prostate cancer. (Bismar et al., NEOPLASIA8(1):59-88 (2006).) It should be noted, however, that severallaboratories reported high AR expression, amplification, or activity inlate stage metastatic prostate cancer. (Heinlein et al., ENDOCRINE REV25:276-308 (2004); Chen et al., NAT MED 10:26-7 (2004) Dehm et al., JCELL BIOCHEM 99:333-344 (2006); Linja et al., CANCER RES 61:3550-55(2001); Li et al., AM J SURG P ATHOL 28:928-34 (2004).) These differentfindings underline the heterogeneous nature of late stage, especiallyandrogen independent, metastatic prostate cancer. (Shah et al., CANCERRES. 64(24):9209-16 (2004).)

To develop an in vivo readout of AR functional status in prostate cancercells, we have been pursuing parallel quantitative measurements ofvarious AR regulated genes in carefully isolated benign and tumor cellsof over 200 specimens as shown in Example 8. Quantitative expressionanalyses of androgen regulated genes at the mRNA level, such asPSA/KLK3, PMEPA1, PCA3, as well as androgen independent genes (AMACR,LTF), representing over 2000 data points, suggest that PSA/KLK3 andother androgen regulated genes reflect in vivo functional status of theAR and that their expression levels can be used to measure positive ornegative correlation with aggressiveness of prostate cancer, as defined,for example, by Gleason grade, pathological stage, and/or biochemicalrecurrence. Initially we chose to focus on PSA/KLK3 mRNA as it is one ofthe most robust direct transcriptional targets of AR and is easilydetectable in prostate cancer cells. (Kim et al., J CELL BIOCHEM93(2):233-41 (2004).)

Our most recent data show that quantitative gene expression patterns ofa panel of AR regulated genes in primary prostate cancer provideprognostic fingerprints. Using high-throughput assays as well asrational candidate gene strategies, we defined a set of six androgen ingenes (PSA/KLK3, PMEPA1, NKX3.1, ODC1, AMD1, and ERG). Differentcombinations of two or more of these six genes, or their isoforms, canbe used to provide a quantitative measure of in vivo AR function inprostate cancer specimens, i.e., the androgen receptor function index,or ARF index (ARFI). Although real time, quantitative PCR (QRT-PCR) wasused to measure the expression levels of these genes, other techniquesknown in the art, such as immunohistochemistry, can be used to detectRNA or protein levels.

The ARFI readout can be converted into a single number indexrepresenting the overall in vivo AR activity, which in turn can beincorporated into nomograms, such as the one created by Kattan et al.,that demonstrated the importance of PSA, Gleason sum, extra capsularextension, surgical margins, seminal vesicle invasion, lymph nodeinvolvement, treatment year, and adjuvant radiotherapy in predicting10-year probability of prostate cancer recurrence after radicalprostatectomy. The nomograms can be used to model time-to-event data,including prediction of prostate cancer progression, combined withestablished clinical and pathological characteristics that predict thisendpoint. The concordance index, C, can be used to assess theimprovement in model fit upon inclusion of ARFI. (Harrell et al., JAMA247(18):2543-6 (1982).) Current nomogram calculators incorporatemeasurable patient factors in an attempt to use such factors to predictan outcome, such as PSA recurrence following surgery, to aid intreatment decision making in advance of invasive procedures.

The ARFI genes are either direct targets of AR or are tightly regulatedby AR, and cover major biological functions regulated by AR in prostatecancer. The gene set includes five androgen regulated genes and ERG. Ouroriginal observations of frequent overexpression of certain isoforms ofERG in prostate cancer (Foleye et al., ENDOCR RELAT CANCER 11(477-88(2004)), and subsequent independent study showing prevalent chromosomalrearrangements leading to the activation of ERG expression throughAR-regulated TMPRSS2 gene promoter (Tomlins et al., SCIENCE310(5748):644-8 (2005)), have highlighted ERG as an aberrant ARactivated gene specific to prostate cancer. Therefore, the quantitativeevaluation of ERG expression has been integrated in ARFI. The ERGread-out can be applied to TMPRSS2-ERG positive tumors, which accountfor greater than 60% of prostate cancer patients. (Id.)

It should be noted that although the following Examples use ERG1 as amodel ERG isoform, ERG2, ERG3, ERG8, EPC1, EPC2, and combinations ofthose ERG isoforms can also be used, as may transcript products from theprostate cancer-specific promoter described in Example 5. Accordingly,any mention of “ERG” in the context of an ARFI readout includes not onlyERG1, but also ERG2, ERG3, ERG8, EPC1, EPC2, and transcript productsfrom the prostate cancer-specific promoter described herein, as well astheir combinations, unless specifically indicated to the contrary bycontext or by an explicit exclusion of one or more of those isoforms.For example, ARFI readouts may employ an ERG gene that is not ERG1 orERG2. Similarly, in some embodiments it may be desirable to includeERG8, EPC1, or EPC2 in the readout, but not ERG1 or ERG2.

EXAMPLE 9 Co-Regulation of ARF1 Genes Reflects Robust in Vivo FunctionalLinkage to AR Signaling

We have recently completed a comprehensive gene expression analyses ofmicrodissected prostate cancer cells and matched benign epithelial cellsfrom radical prostatectomy specimens of 40 patients (80 Gene hips)(Petrovics et al., ONCOGENE 24:3847-52 (2005)). The GeneChip dataset wasevaluated for androgen regulated gene expression, PSA/KLK3, PMEPA1NKX3.1, ODC1, and AMD1, along with ERG (which can become androgenregulated in prostate cancer cells through fusion with a TMPRSS2promoter in the majority of patients), were selected by their widedynamic ranges of expression, as well as by their reported response toandrogenic stimuli. (Heinlein et al., ENDOCRINE REV 25:276-308 (2004);Linja et al., J STEROD BIOCHEM MOL BIOL 92:255-64 (2004); Shaffer et alLANCET ONCOL 4:407-14 (2003); Chen et al., NAT MED 10:26-7 (2004); Dehmet al., J CELL BIOCHEM 99:333-344 (2006); Segawa et al., ONCOGENE21(57):8749-58 (2002); Xu et al., INT J CANCER 92(3):322-8 (2001).)Moreover, some of these genes (NKX3.1, ERG, PMEPA1) may be causallylinked to prostate cancer development.

The concerted expression of this gene panel (ARFI) is reflective of thefunctional status of in vivo AR activity. Normalized expressionintensity values are depicted in a heat map format (FIG. 12). Anon-supervised hierarchical cluster analysis (software from TIGR,Gaithersburg, Md.) was performed both by patients and also by genes andrevealed robust in vivo co-regulation of ARFI genes in the tumor cellsof prostate cancer patients, reflecting either active or dysfunctionalAR (FIG. 12). Two tight gene sub clusters emerged: PSA/KLK3, NKX3.1,PMEPA1, and ODC1, AMD1 (polyamine pathway), differing in expression onlyin the middle 12-patient cluster, which underlines the importance ofusing a panel of ARFI genes representing different downstream ARpathways. The other two large patient clusters show tight co-regulationof all ARFI genes reflecting either active AR (left 17-patient cluster),or dysfunctional AR (right 11-patient cluster) (FIG. 12). ERG alsoco-regulates closely with other ARFI genes in tumor cells of themajority of prostate cancer patients, where ERG is likely fused to theandrogen regulated TMPRSS2 promoter, providing a highly specific tumorcell marker.

We have also shown that ERG can be used as part of a multigene panelwith other prostate cancer-associated genes that are not androgenregulated. FIG. 13 shows a heat map for a multigene panel that includesERG, AMACR, DD3, PSGR, and PCGEM1, The heat map is a non-supervisedhierarchical clustering of tumor over normal gene expression ratiosderived from TagMan QRT-PCR analysis of microdissected cell samples fromprostate tissue sections. When non-AR genes were used in the multigenepanel, we found strong overexpression of the various marker genes indistinct, but overlapping subsets of patients.

EXAMPLE 10 Validation of in Vivo Co-Regulation of ARFI Genes by QRT-PCR

Using QRT-PCR, we evaluated the expression of ERG transcripts for arelationship with the expression of the androgen-regulated genes,PSA/KLK3 and PMEPA1 (Dehm et al., J CELL BIOCHEM 99:333-344 (2006); Xuet al., CANCER RES 63(15):4299-304 (2003)), in prostate cancer cells ofpatients with TMPRSS2-ERG fusion. LTF (Ward et al., CELL MOL LIFE SCI62(22):2540-8 (2005)), a non-androgen regulated control gene, wasassayed in the same tumor cells (FIG. 14). In the figure, significantcorrelations (R>0.5) are marked by solid bars. LTF, a non-androgenregulated gene, was used as a negative control. The Pearson correlationcoefficient (R) is shown above the bars. P values and the number ofpatients (n) assessed in the experiments are indicated under the bars.

Sixty five patients with detectable TMPRSS2-ERG fusion transcript inprostate cancer cells were selected for this study. Strikingco-regulation was observed between the expression levels of ERG, tissuePSA/KLK3 (p<0.0001) and PMEPA1 (p<0.0001) in patients with detectableTMPRSS2-ERG transcripts. The co-regulation is even stronger in thesubset of these patients where the expression level of the TMPRSS2-ERGfusion transcript is above the median (“High fusion transcript,” FIG. 14right panel). These data indicate that the level of co-regulation withinthe ARFI genes (including TMPRSS2-ERG) reflects the overall functionalstatus of AR in prostate cancer cells and that decreased expression ofARFI genes correlates with compromised or diminished androgen receptorsignaling in prostate tumor cells. Furthermore, the data indicate thatthe expression levels of ARFI genes are reduced in advanced prostatecancer, such as pT3 stage prostate cancer.

EXAMPLE 11 PSA/KLK3 and TMPRSS2-ERG Indicate a Decrease of in Vivo ARActivity During Prostate Cancer Progression

PSA/KLK3 and ERG mRNA expression were further analyzed for theirrelationship to prostate cancer progression in a larger patient cohort.As shown in FIG. 15, patients with pT3 prostate cancer (locally invasivetumor growing outside the capsule) had significantly (p=0.0098) lowerexpression of PSA/KLK3 transcript levels as compared to patients withpT2 stage disease (organ confined). Moreover, decreased TMPRSS2-ERGfusion transcript levels were also apparent in the prostate cancer cellsof pT3 patients (p=0.0275).

To study patients with intermediate serum PSA levels, further analysiswas limited to patients with serum PSA from 2 to 10 ng/mL (n=79). Basedon serum PSA levels, these patients have an uncertain prognosis. FIG. 16shows the distribution of PSA/KLK3 mRNA expression levels in tumor cellsof prostate cancer patients with biochemical recurrence.

Statistical analysis of the data presented in FIG. 16 demonstrates thatthe expression of tissue PSA/KLK3 mRNA in tumor cells of biochemicalrecurrence free patients was significantly higher than in patients withbiochemical recurrence (p=0.0062, Student t-test). PSA/KLK3 mRNAexpression in benign epithelial cells did not show such correlation.This prostate cancer patient cohort was divided into quintiles based ontissue PSA/KLK3 mRNA expression levels in tumor cells, and was comparedwith respect to time to biochemical relapse. As seen in FIG. 17, anunadjusted Kaplan-Meier analysis demonstrates improved biochemicalsurvival for patients with the highest tissue PSA/KLK3 mRNA expression(Quintiles 1 and 2) (p=0.0229). Thus, PSA/KLK3 mRNA expression in tumorcells of prostate cancer patients inversely correlates with diseaserecurrence. High expression levels of tumor PSA/KLK3 mRNA correlateswith biochemical recurrence free survival, whereas with low expressionlevels of PSA/KLK3 mRNA reflect an alteration of AR signaling in thetumor cell microenvironment, leading to an increased likelihood of tumorrecurrence after prostatectomy.

EXAMPLE 12 ERG Activates C-MYC, a Central Target of ERG in ProstateCancer

Inhibiting ERG decreased CMYC expression and upregulated the prostatedifferentiation marker genes PSA and prostein. Furthermore, inhibitingC-MYC recapitulated the ERG siRNA phenotype. A double knockdown of ERGand C-MYC using 50-50% doses of inhibitory ERG and C-MYC siRNA moleculeseffectively controlled cell growth and rescued the differentiationprogram in prostate cancer cells (FIG. 18). C-MYC expressionsignificantly correlated with ERG expression in human prostate tumorcells (FIG. 18).

Small interfering RNA (“siRNA”) oligo duplexes designed to interferewith ERG function were based on the human ERG having the NCBI locus IDGXL_163565 and Accession No. NM_004440. The ERG siRNA5′-CGACATCCTTCTCTCACAT-3′ (SEQ ID NO: 29) was purchased from Dharmacon,Lafayette, Colo.). The siRNA pool against human C-MYC (L-003282-00)(Locus ID: GXL_07312, Accession: NM_002467) and non-target (“NT”) siRNAduplexes (D-001206-13-20) were both from Dharmacon, Lafayette, Colo.

VCaP cells were seeded into 10 cm tissue culture dishes in mediumcontaining 10% charcoal-treated fetal bovine serum (“cFBS”) (GeminiBio-Products, Calabasas, Calif.) for three days. The cells weretransfected with 50 nM NT, 50 nM ERG siRNA, 50 nM MYC siRNA, or thecombination of 25 nM ERG siRNA and 25 nM MYC siRNA. Cotransfection ofsiRNAs and plasmids was carried out with Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.), as described by the manufacturer for HEK 293 cells.

The coding region of ERG (NM_001440) was sub-cloned into an adenoviraltransfer vector containing an internal ribosome entry site (“IRES”),wherein green fluorescent protein (“GFP”) is expressed from the IREStranslation initiation sequence (“IRES-GFP”). The generation ofrecombinant adenovirus plasmid and production of recombinant adenoviruswere performed as described by Sun et al., ONCOGENE 25, 3905-13 (2006).Adenovirus titer was determined by GFP assay and plaque forming assay.VCaP and LNCaP cells were infected with the Ad-ERG or Ad-Control vectorsand the proteins were detected by Western blot. A wild type ERG3 (NCBIAccession No. NM_182918) expression vector (pIRES-EGFP-ERG3) wasgenerated by amplifying the coding sequences with the primers6-GGCTTTGATGAAAGCTCTAAACAAC-3′ (SEQ ID NO: 50) andTCAAAAGTGCCTCAAGAGGA-3′ (SEQ ID NO: 51) from a human normal prostatecDNA library (Catalog No. AM 3337, Ambion, Austin, Tex.) and wasverified by DNA sequencing. HEK293 cells were transfected with wild typeERG3 or TM-ERG3-expressing vectors and the proteins were detected byWestern blot.

Twelve hours after transfection with siRNA, the cells were treated with100 pM R1881 and processed for the subsequent analyses. To knockdownectopic ERG protein, TMPRSS2-ERG2 and TMPRSS2-ERG8 plasmids (pIRES-EGFP,Clontech, Palo Alto, Calif.) were generated by PCR amplification ofhuman TMPRESS2 and ERG cDNA with the primers 5′-TAGGCGCGAGCTAAGCAGGAG-3′(SEQ ID NO: 8) and 5′-CCCTCCCAAGAGTCTTTGGATCTC-3′ (SEQ ID NO: 12). Thesequences were verified by DNA sequencing. Cotransfection of siRNAs andplasmids was carried out with Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.), as described by the manufacturer for HEK 293 cells.

C-MYC expression in response to inhibition by ERG siRNA was measured byQRT-PCR and Western blot. In the QRT-PCR analysis, VCaP cells weretransfected with siRNAs and harvested two or four days aftertransfection. Total RNA preparation and RT-PCR were performed asdescribed by Gao et al., CLIN CAN RES 9:2545-50 (2003). Each RNA samplewas evaluated for ERG knockdown by ERG siRNA in triplicate RT-PCRreactions and one control reaction performed in the absence of reversetranscriptase. The ERG PCR forward primer 5′-ACCGTTGGGATGAACTACGGCA-3′(SEQ ID NO: 10) and reverse primer 5′-TGGAGATGTGAGAGAAGGATGTCG-3′ (SEQID NO: 53) were used in the reaction. GAPDH gene expression was detectedusing forward primer 5′-GAGCCACATCGCCTCAGACACC-3′ (SEQ ID NO: 54) andreverse primer GTTCTCAGCTTGACGGTGCC-3′ (SEQ ID NO: 55). RT-PCR derivedERG or GAPDH fragments were separated by electrophoresis on Tris-borateEDTA-1% agarose gels and visualized by ethidium bromide staining. Banddensities were quantified with Quantity One (Bio-Rad Laboratories,Hercules, Calif.) and ERG expression was normalized to GAPDH levels.

In the Western blot analysis, cells were lysed in M-PER mammalianprotein extraction reagent (Pierce, Rockford, Ill.) supplemented withprotease and phosphatase inhibitor cocktails (Sigma, St. Louis, Mo.).Immunoblot assays were performed according to standard procedures, forexample, probing NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, Calif.)with antibodies. Antibodies used in Western blot experiments include animmunoaffinity purified anti-ERG peptide polyclonal antibody prepared inour laboratory to the peptide having the amino acid sequenceDFHGIAQALQPHPPESSLYKYPSDLPYMGSYHAHPQKMNFVAPHPPAL (SEQ ID NO: 52),anti-PSA (Deka, Carpinteria, Calif.), anti-MYC (Upstate Biotechnology,Lake Placid, N.Y.), anti-SLC45A3 (Dako, Carpinteria, Calif.) oranti-GAPDH antibodies (SantaCruz, Santa Cruz, Calif.). To detect theendogenous ERG protein in VCaP cells, 80 μg cell lysate were loaded intoeach lane of the gel.

Reduced recruitment of ERG to the MYC P2 promoter downstream ETS elementwas assessed at 48 hours post-transfection by chromatinimmunoprecipitation (“ChIP”) assay, using an anti-ERG antibody (FIG. 18,bottom left panel). IgG and control genomic DNA amplicons (“Input”) wereused as controls.

ChIP assays were performed according to Masuda et al., J MOL BIOL353:763-71 (2005). To detect specific ChIP products, 38 amplificationcycles were performed. ETS binding sites within the target regions wereidentified by matrix match analysis using the GEMS Launcher software(Genomatix GmbH, Munich, Germany). ERG protein was detected with thepolyclonal anti-ERG antibody sc-363 (SantaCruz Biotechnology, Inc.,Santa Cruz, Calif.). Promoters directed to NCBI Accession No.GLX_573121NM_002467 amplified the human C-MYC gene. The primer pairdirected to the ETS binding site V$EISF/PDEF0.1 P2 downstream promoter5′-GCCCCTTGCATCCTGAGCTCC-3′ (SEQ ID NO: 56) directed the5′-GGTCGGACATTCCTGCTTTA-3′ (SEQ ID NO: 57) and5′-ACCCAACACCACGTCCTAAC-3′ (SEQ ID NO: 58) was used as described inMeulia et al., MOL CELL BIOL 12:4590-600 were used. The androgenreceptor (“AR”) was immunoprecipitated with anti-AR antibodies and thePSA(KLK3) AREIII enhancer target region was amplified as described.(Masuda et al., J MOL B IOL 353:763-71 (2005)). The SLC45A3 gene (NCBIAccession No. GXL_151340/XM_001490454) promoter upstream ARE(V$GREF_ARE0.2) and ETS (V$ETSF_PDEF0.1) binding site containing region(5′-AGAGCACAGAAAGGCTGCCCTGG AAGTGGCTGGGCATCC TGTCAGCT-3) (SEQ ID NO: 59)was amplified by the 5′-TGTGGGACTTCTCTGC TGAA-3′ (SEQ ID NO: 60) and5′-CAACGTTCAAGGGGAAGAAA-3′ (SEQ ID NO. 61) primers.

Cell morphology was also examined. Photomicrographs of VCaP cells at day8 are shown in the top right panel of FIG. 18. PSA mRNA and proteinexpression were also measured by QRT-PCR and Western blot assays,respectively, and are shown in the bottom panel of FIG. 18. Cell lysateswere prepared and assayed by Western blot with anti-C-MYC, anti-PSA, oranti-SLC45A3 (prostein) antibodies. Tubulin was used as a control.

ERG-MYC correlation analysis was performed by assessing quantitativegene expression data of C-MYC and ERG or C-MYC and PCA3 from 37 lasercapture microdissected human tumors. The results were expressed as R andP-values, as shown in the table in the bottom right panel of FIG. 18.The correlation of ERG with C-MYC is highly significant; R=05548,p=0.0004.

EXAMPLE 13 GeneChip® Data Analysis

VCaP cells were plated in medium containing 10% cFBS (GeminiBio-Products, Calabasas, Calif.) for three days. Cells were transfectedwith 50 nM of siERG or 50 nM of control NT and grown in FBS containingmedia for 24 or 48 hours. Total RNA was isolated, five micrograms of RNAfrom each of the VCaP transfectants were biotin labeled, and GeneChip®HG U133 Plus 2.0 chips were hybridized with the labeled probes.Expression data were normalized by Robust Multi-array Averages (RMA) andfold changes in ERGsi/NT (24 h) and ERGsi/NT (48 h) treatment groupswere calculated. A two fold cut-off criterion was applied for subsequentpathway analysis by the Bibliosphere Software using the functionalco-citation-based analysis function (Genomatix GmbH, Munich, Germany)(Scherf et al., BRIEF BIOINFORM 6:287-97 (2005). Total RNA from humanprostate specimens was isolated from laser capture microdissected tumorand benign epithelial cells, as described by Shaheduzzamen et al., CANBIOL THER 6:(2007, Epub ahead of print). The RNA was quantified,amplified, biotinylated, hybridized to the high-density oligonucleotidehuman genome array HG U1133A (Affymetrix, Santa Clara, Calif.), andnormalized as described by Petrovics et al., ONCOGENE 24:3847-52 (2005).Tumor/normal ERG expression ratios were evaluated in the data set fromwell-differentiated prostate tumors. Gene expression changes wereaveraged in seven data sets where ERG expression was elevated 19-38fold. A two-fold cut-off criterion was applied for further pathwayanalysis using Bibliosphere Software (Genomatix GmbH, Munich, Germany).Normalized human ERG overexpressing tumor data was compared to the 48 hERG siRNA treatment gene expression data. Common genes were selected forthe subsequent network analysis by the Bibliosphere Software.

FIG. 20 shows a gene network in ERG expressing human prostate tumors.Normalized (tumor/normal) gene expression data of sevenwell-differentiated prostate tumors with 19-38 folds of ERGoverexpression were analyzed by the Bibliosphere software (Shaheduzzamenet al., CAN BIOL THER 6:(2007, Epub ahead of print)). Red (medium grey)and yellow (light grey) boxes indicate upregulation, shades of blue(dark grey) indicate downregulation. The functional connections aredisclosed in the insert to FIG. 20.

The network of genes affected in response to ERG knockdown in VCaP cellsis shown in FIG. 21. Cells were transfected with either 50 nM of ERGsiRNA or with 50 nM of NT and were incubated for 24 hours (left sidecodes) or 48 hour (right side codes). For probe labeling, total RNA wasisolated from the cells and was labeled for hybridization. GeneChip® HGU133 Plus 2.0 chips were hybridized with the labeled probes. ERG si/NTexpression ratios were calculated and a two-fold cut-off criterion wasapplied towards the subsequent pathway analysis by Bibliosphere SoftwareGene symbols within the boxes indicate changes in gene expression (FIG.21).

Transient ERG expression diminished PSA protein levels and decreasedrecruitment of AR to the PSA AREIII enhancer. VCaP and LNCaP cells wereinfected with adenoviral ERG (Ad-ERG) or Ad-Control (Control) vectors.Cell lysates prepared at 24, 48, and 72 hours post-infection wereanalyzed in immunoblot assays with anti-ERG, anti-PSA, and anti-tubulinantibodies. ChiP assessment of AR recruitment to the KLK3/PSA geneAREIII enhancer in VCaP and LNCaP cells in response to the transientexpression of ERG by adenoviral Ad-ERG or Control vectors (FIG. 22).

EXAMPLE 14 Prostate Differentiation Genes are Repressed by ERG

ERG siRNA treatment resulted in the recruitment of androgen receptor tothe PSA AREIII enhancer (FIG. 23). PSA mRNA and protein expression weremeasured by QRT-PCR and Western blot assays, respectively. Increased ARbinding to the PSA enhancer (ARE) and decreased ERG recruitment to theoverlapping ETS cognate element was measured by ChIP assay 48 hoursafter transfection. After 9 days of ERG siRNA treatment, VCaP cells werestained for cytokeratin CK8/18, PSA, and DNA (FIG. 23).

ERG siRNA treatment also resulted in the recruitment of androgenreceptor to the prostein (SLC45A3) gene upstream enhancer. Expression ofTMPRSS2-ERG negatively correlated with prostein (SLC45A3) expression inhuman specimens (FIG. 24). SLC45A3 (Prostein) expression in ERG siRNAtransfected VCaP cells was measured by Western blot. Recruitment of ARand ERG to the SLC45A3 promoter upstream ARE and ETS elements wasassessed by ChIP assay 48 hours post-transfection. A matrixrepresentation of TMPRSS2-ERG expression and SLC45A3 immunostaining ofprostate tumors of 26 patients demonstrated a correlation between ERGsiRNA treatment and prostein expression. Sections of whole mountedradical prostatectomy specimens were assessed by immunohistochemistrywith anti-SLC45A3 (SLC) antibody.

Radical prostatectomy specimens from 26 patients were fixed in 10%buffered formalin and embedded as whole mounts in paraffin. Eachprostate was sectioned at 0.22 cm intervals in a transverse planeperpendicular to the long axis of the posterior surface of the prostateand completely embedded as whole mounts. The volume of each tumor wascalculated in three dimensions (apex to base, right to left, andanterior to posterior) using the largest dimension in each direction todetermine the index tumor. Index tumor was analyzed for the presence orabsence of TMPRSS2-ERG fusion transcripts as described by Furusato etal., MOD PATHOL 21(2):67-75 (2008), and for Prostein immunohistochemicalstaining on adjacent four-micron sections of the whole-mounted blocks.Slides were incubated with anti-SLC45A3 antibody (Dako North America,Carpinteria, Calif.), diluted 1:160. Vector VIP (purple) was used as thechromogen substrate (Vector Laboratories, Burlingame, Calif.) and theslides were counterstained with hematoxylin. SLC45A3 expression wasassessed based on both the amount and intensity of immunopositive cells.Intensities were scored as “0” if not stained, “1” if stained weakly,and “2” if stained strongly. The percentage of positively stained areawas also estimated and scored as “1” if less than 25% of the areastained positive, “2” if 25-50% stained positive, “3” if 51-75% stainedpositive, and “4” if more than 75% stained positive. The final score wasdetermined by multiplying the intensity score and the percentage ofpositively stained area.

VCaP cells were fixed with 4% paraformaldehyde and centrifuged ontosilanized slides (Sigma, St. Louis, Mo.) with a cytospin centrifuge.Cells were immunostained with anti-cytokeratin 8/18 and anti-PSA (bothfrom Delco, Carpinteria, Calif.) followed by goat anti-mouse Alexa-488and goat anti-rabbit Alexa-594 secondary antibodies (Invitrogen,Carlsbad, Calif.). Images were captured using a 40×/0.65 N-Planobjective lens on a Leica DMLB upright microscope with a QImagingRetiga-EX CCD camera (Burnaby, BC, Canada) controlled by OpenLabsoftware (Improvision, Lexington, Mass.). Images were converted intocolor and merged by using Adobe Photoshop.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan readily recognizes that many other embodiments areencompassed by the invention.

1-11. (canceled)
 12. An isolated nucleic acid molecule comprising SEQ IDNO: 46, a biologically active fragment thereof, or the complementthereof. 13-20. (canceled)
 21. A method of treating prostate cancercomprising destabilizing a prostate cancer-specific ERG gene transcriptin prostate cancer cells. 22-27. (canceled)
 28. The method of claim 21,wherein the prostate cancer-specific ERG gene transcript comprisesnucleotides 843-861 of SEQ ID NO:
 46. 29. The method of claim 21,wherein the prostate cancer-specific ERG gene transcript comprisesnucleotides 970-988 of SEQ ID NO:
 46. 30. The method of claim 21,wherein the prostate cancer-specific ERG gene transcript comprisesnucleotides 1261-1279 of SEQ ID NO:
 46. 31. The method of claim 21,wherein the prostate cancer-specific ERG gene transcript comprisesnucleotides 1398-1416 of SEQ ID NO:
 46. 32. The method of claim 21,wherein the prostate cancer-specific ERG gene transcript comprisesnucleotides 1581-1599 of SEQ ID NO:
 46. 33. The method of claim 21,wherein the destabilization comprises administering an interfering RNA.34. The method of any of claim 21, wherein the destabilization comprisesadministering an antisense nucleic acid. 35-43. (canceled)
 44. Anisolated nucleic acid molecule, comprising at least about 15 contiguousnucleotides of SEQ ID NO: 46, wherein the nucleic acid is capable ofhybridizing to SEQ ID NO: 46, or the complement thereof, underconditions of high stringency but not to ERG1, ERG2, ERG3, ERG4, ERG5,ERG6, ERG7, ERG9, EPC1, EPC2, or TMPRSS2.
 45. The isolated nucleic acidof claim 44, wherein the nucleic acid is up to about 50 nucleotideslong.
 46. The isolated nucleic acid of claim 44, wherein the conditionsof high stringency comprise hybridization for 12 hours at 65° C. in6×SSC followed by a wash in 0.1×SSC at 50° C. for 45 minutes. 47-57.(canceled)